Peptides and combination thereof for use in the immunotherapy against cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/949,665, filed Apr. 10, 2018, which claimsbenefit to U.S. Provisional Application No. 62/483,702, filed Apr. 10,2017, and is related to German Application No. 102017107697.2, filedApr. 10, 2017, the content of which is incorporated herein by referencein its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2332.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-085002_ST25.txt” createdon Feb. 18, 2020 and 41,792 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference. Pursuant to the EFS-Weblegal framework and 37 CFR §§ 1.821-825 (see MPEP § 2332.03(a)), aSequence Listing in the form of an ASCII-compliant text file (entitled“Substitute Sequence_Listing_2912919-085002_ST25.txt” created on May 27,2020 and 83,071 bytes in size) is submitted concurrently with theresponse to the Non-Final Office Action filed on Aug. 10, 2020, and theentire contents of the Substitute Sequence Listing are incorporatedherein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

According to the World Health Organization (WHO), cancer ranged amongthe four major non-communicable deadly diseases worldwide in 2012. Forthe same year, colorectal cancer, breast cancer and respiratory tractcancers were listed within the top 10 causes of death in high incomecountries.

In 2012, 14.1 million new cancer cases, 32.6 million patients sufferingfrom cancer (within 5 years of diagnosis) and 8.2 million cancer deathswere estimated worldwide (Ferlay et al., 2013; Bray et al., 2013).

Estimated incidences of different cancer types (adult population, bothsexes) world-wide in 2012 were (Ferlay et al., 2013; Bray et al., 2013):For cancer of the brain and nervous system: 256213, for colorectalcancer: 1360602, for kidney cancer: 337860, for liver cancer: 782451,and for gastric cancer: 951594 cases.

Estimated incidences of different cancer types (adult population, bothsexes) in the USA, EU-28, China and Japan in 2012 were (Ferlay et al.,2013; Bray et al., 2013): For cancer of the brain and nervous system:135884, for colorectal cancer: 845797, for kidney cancer: 226733, forliver cancer: 513172, and for gastric cancer: 615641 cases.

Estimated mortalities of different cancer types (adult population, bothsexes) world-wide in 2012 were (Ferlay et al., 2013; Bray et al., 2013):For cancer of the brain and nervous system: 189382, for colorectalcancer: 693933, for kidney cancer: 143406, for liver cancer: 745533, andfor gastric cancer: 723073 cases.

Estimated mortalities of different cancer types (adult population, bothsexes) in the USA, EU-28, China and Japan in 2012 were (Ferlay et al.,2013; Bray et al., 2013): For cancer of the brain and nervous system:100865, for colorectal cancer: 396066, for kidney cancer: 83741, forliver cancer: 488485, and for gastric cancer: 447735 cases.

Within the groups of brain cancer, the current invention specificallyfocuses on glioblastoma (GBM). GBM is the most common central nervoussystem malignancy with an age-adjusted incidence rate of 3.19 per100,000 inhabitants within the United States. GBM has a very poorprognosis with a 1-year survival rate of 35% and a 5-year survival ratelower than 5%. Male gender, older age and ethnicity appear to be riskfactors for GBM (Thakkar et al., 2014).

Colorectal cancer—Depending on the colorectal cancer (CRC) stage,different standard therapies are available for colon and rectal cancer.Standard procedures include surgery, radiation therapy, chemotherapy andtargeted therapy for CRC (Berman et al., 2015a; Berman et al., 2015b).

Removal of the tumor is essential for the treatment of CRC. Anatomicconditions differ for rectal carcinomas from another CRC as the rectumis located in the pelvis and the tumor can be difficult to access.Well-differentiated small rectal tumors (stage T1) require excision, butno further treatment with chemotherapy. Patients with rectal tumors ofhigher T stages receive neoadjuvant radio-chemotherapy with afluoropyrimidine prior to total mesorectal excision (TME) and adjuvantchemotherapy. For chemotherapeutic treatment, the drugs capecitabine or5-fluorouracil (5-FU) are used. For combinational chemotherapy, acocktail containing 5-FU, leucovorin and oxaliplatin (FOLFOX) isrecommended (Stintzing, 2014; Berman et al., 2015b).

Treatment of colon carcinomas involves radical hemicolectomy and lymphnode resection. Early stages (UICC stage I) do not require additionaltreatment. Patients with tumors of UICC stage II receive 5-FU orcapecitabine. Treatment for patients with UICC stage III includes thedrug combinations FOLFOX and XELOX (capecitabine plus oxaliplatin)(Berman et al., 2015a; Stintzing, 2014).

Metastatic, unresectable CRC are treated with chemotherapeuticalcocktails such as FOLFIRI (5-FU, leucovorin, irinotecan), FOLFOX,FOLFOXIRI (5-FU, irinotecan, oxaliplatin), FOLFOX/capecitabine,FOLFOX/oxaliplatin, FOLFIRI/capecitabine and irinotecan or UFT (5-FU,tegafur-uracil) (Stintzing, 2014).

In addition to chemotherapeutic drugs, several monoclonal antibodiestargeting the epidermal growth factor receptor (EGFR, cetuximab,panitumumab) or the vascular endothelial growth factor-A (VEGF-A,bevacizumab) are administered to patients with high stage disease. Forsecond-line and later treatment the inhibitor for VEGF aflibercept, thetyrosine kinase inhibitor regorafenib and the thymidylate-synthetaseinhibitor TAS-102 and the dUTPase inhibitor TAS-114 can be used(Stintzing, 2014; Wilson et al., 2014).

Latest clinical trials analyze active immunotherapy as a treatmentoption against CRC. Those strategies include the vaccination withpeptides from tumor-associated antigens (TAAs), whole tumor cells,dendritic cell (DC) vaccines and viral vectors (Koido et al., 2013).

Peptide vaccines have so far been directed against carcinoembryonicantigen (CEA), mucin 1, EGFR, squamous cell carcinoma antigen recognizedby T cells 3 (SART3), beta-human chorionic gonadotropin (beta-hCG),Wilms' Tumor antigen 1 (WT1), Survivin-2B, MAGE3, p53, ring fingerprotein 43 and translocase of the outer mitochondrial membrane 34(TOMM34), or mutated KRAS. In several phase I and II clinical trialspatients showed antigen-specific CTL responses or antibody production.In contrast to immunological responses, many patients did not benefitfrom peptide vaccines on the clinical level (Koido et al., 2013; Miyagiet al., 2001; Moulton et al., 2002; Okuno et al., 2011).

Dendritic cell vaccines comprise DCs pulsed with either TAA-derivedpeptides, tumor cell lysates, apoptotic tumor cells, or tumor RNA orDC-tumor cell fusion products. While many patients in phase I/II trialsshowed specific immunological responses, only the minority had aclinical benefit (Koido et al., 2013).

Whole tumor cell vaccines consist of autologous tumor cells modified tosecrete GM-CSF, modified by irradiation or virus-infected, irradiatedcells. Most patients showed no clinical benefit in several phase II/IIItrials (Koido et al., 2013).

Vaccinia virus or replication-defective avian poxvirus encoding CEA aswell as B7.1, ICAM-1 and LFA-3 have been used as vehicles in viralvector vaccines in phase I clinical trials. A different study usednon-replicating canary pox virus encoding CEA and B7.1. Besides theinduction of CEA-specific T cell responses 40% of patients showedobjective clinical responses (Horig et al., 2000; Kaufman et al., 2008).

Gastric cancer—The wall of the stomach is made up of 3 layers of tissue:the mucosal (innermost) layer, the muscularis (middle) layer, and theserosal (outermost) layer. Gastric cancer (GC) begins in the cellslining the mucosal layer and spreads through the outer layers as itgrows. Four types of standard treatment are used. Treatment for gastriccancer may involve endoscopic or surgical resection, chemotherapy,radiation therapy or chemoradiation. Surgery is the primary treatmentand the only curative treatment for gastric cancer. Since the earlystages of gastric cancer are mostly asymptomatic, the disease is usuallydiagnosed in an advanced stage. For metastatic gastric cancer, noglobally accepted standard chemotherapy combination regimen has yet beenestablished. However, the combination of 5-FU and a platinum analog isstill the most widely accepted reference regimen worldwide, although5-FU can be replaced by capecitabine or irinotecan and cisplatin can bereplaced by oxaliplatin. Additionally, triple-combination therapiescomprising cisplatin, 5-FU and docetaxel or, in the case of HER-2over-expressing tumors, cisplatin, 5-FU and trastuzumab can be applied(Leitlinie Magenkarzinom, 2012).

The efficacy of current therapeutic regimens for advanced GC is poor,resulting in low 5-year survival rates. Immunotherapy might be analternative approach to ameliorate the survival of GC patients. Adoptivetransfer of tumor-associated lymphocytes and cytokine induced killercells, peptide-based vaccines targeting HER2/neu, MAGE-3 or vascularendothelial growth factor receptor 1 and 2 and dendritic cell-basedvaccines targeting HER2/neu showed promising results in clinical GCtrials. Immune checkpoint inhibition and engineered T cells mightrepresent additional therapeutic options, which is currently evaluatedin pre-clinical and clinical studies (Matsueda and Graham, 2014).

Glioblastoma—The therapeutic options for glioblastoma (WHO grade IV) arevery limited. According to the guidelines released by the German Societyfor Neurology the standard therapy in young patients includes resectionor biopsy of the tumor, focal radiation therapy and chemotherapy withtemozolomide. Alternative chemotherapeutic regimens consist ofCCNU/Iomustine or a combination of procarbazine with CCNU andvincristine (PCV). In elderly patients' resection or biopsy of the tumorare not recommended. These patients receive chemo- or radiation therapy,depending on the methylation state of theO6-methylguanine-DNA-methyltransferase-(MGMT)-promotor. Negativemethylation state is an indication for focal radiation therapy, whereaspositive methylation state is an indication for temozolomide treatmentwith or without focal radiation therapy. Relapse therapy comprises againresection as well as chemo- and radiation therapy. In the USA, Canadaand Switzerland treatment with bevacizumab (anti-VEGF-antibody) is alsoapproved for relapse therapy (Leitlinien für Diagnostik und Therapie inder Neurologie, 2014).

Different immunotherapeutic approaches are investigated for thetreatment of GB, including immune-checkpoint inhibition, vaccination andadoptive transfer of engineered T cells.

Antibodies directed against inhibitory T cell receptors or their ligandswere shown to efficiently enhance T cell-mediated anti-tumor immuneresponses in different cancer types, including melanoma and bladdercancer. The effects of T cell activating antibodies like ipilimumab andnivolumab are therefore assessed in clinical GB trials, but preliminarydata indicate autoimmune-related adverse events.

Different vaccination strategies for GB patients are currentlyinvestigated, including peptide-based vaccines, heat-shock proteinvaccines, autologous tumor cell vaccines, dendritic cell-based vaccinesand viral protein-based vaccines. In these approaches peptides derivedfrom GB-associated proteins like epidermal growth factor receptorvariant III (EGFRvIII) or heat shock proteins or dendritic cells pulsedwith autologous tumor cell lysate or cytomegalo virus components areapplied to induce an anti-tumor immune response in GB patients. Severalof these studies reveal good safety and tolerability profiles as well aspromising efficacy data.

Adoptive transfer of genetically modified T cells is an additionalimmunotherapeutic approach for the treatment of GB. Different clinicaltrials currently evaluate the safety and efficacy of chimeric antigenreceptor bearing T cells directed against HER2, IL-13 receptor alpha 2and EGFRvIII (Ampie et al., 2015).

Liver cancer—Disease management depends on the tumor stage at the timeof diagnosis and the overall condition of the liver. If possible, partsof the liver (partial hepatectomy) or the whole organ (liver resection)is removed by surgery. Especially patients with small or completelyresectable tumors are qualified to receive a liver transplant.

If surgery is not a treatment option, different other therapies areavailable at hand. For tumor ablation, a probe is injected into theliver and the tumor is destroyed by radio or microwaves or cryotherapy.In embolization procedures, the blood supply of the tumor is blocked bymechanical or chemical means. High energy radio waves can be used todestroy the tumor in radiation therapy.

Chemotherapy against HCC includes combinations of doxorubicin,5-fluorouracil and cisplatin for systemic therapy and doxorubicin,floxuridine and mitomycin C for hepatic artery infusions. However, mostHCC show a high resistance to chemotherapeutics (Enguita-German andFortes, 2014).

Therapeutic options in advanced non-resectable HCC are limited toSorafenib, a multi-tyrosine kinase inhibitor (Chang et al., 2007;Wilhelm et al., 2004). Sorafenib is the only systemic drug confirmed toincrease survival by about 3 months and currently represents the onlyexperimental treatment option for such patients (Chapiro et al., 2014;Llovet et al., 2008).

Lately, a limited number of immunotherapy trials for HCC have beenconducted. Cytokines have been used to activate subsets of immune cellsand/or increase the tumor immunogenicity (Reinisch et al., 2002; Sangroet al., 2004). Other trials have focused on the infusion ofTumor-infiltrating lymphocytes or activated peripheral blood lymphocytes(Shi et al., 2004; Takayama et al., 1991; Takayama et al., 2000).

So far, a small number of therapeutic vaccination trials have beenexecuted. Butterfield et al. conducted two trials using peptides derivedfrom alpha-fetoprotein (AFP) as a vaccine or DCs loaded with AFPpeptides ex vivo (Butterfield et al., 2003; Butterfield et al., 2006).In two different studies, autologous dendritic cells (DCs) were pulsedex vivo with autologous tumor lysate (Lee et al., 2005) or lysate of thehepatoblastoma cell line HepG2 (Palmer et al., 2009). So far,vaccination trials have only shown limited improvements in clinicaloutcomes.

Renal cell carcinoma—Initial treatment is most commonly either partialor complete removal of the affected kidney(s) and remains the mainstayof curative treatment (Rini et al., 2008). For first-line treatment ofpatients with poor prognostic score a guidance elaborated by severalcancer organizations and societies recommend the receptor tyrosinekinase inhibitors (TKIs) sunitinib and pazopanib, the monoclonalantibody bevacizumab combined with interferon-α (IFN-α) and the mTORinhibitor temsirolimus. Based on guidelines elaborated by the US NCCN aswell as the European EAU and ESMO, the TKIs sorafenib, pazopanib orrecently axitinib are recommended as second-line therapy in RCC patientswho have failed prior therapy with cytokines (IFN-α, IL-2). The NCCNguidelines advise also sunitinib in this setting (high-level evidenceaccording to NCCN Category I).

Everolimus and axitinib are recommended as second-line therapy of thosepatients who have not benefited from a VEGF-targeted therapy with TKIsaccording to the established guidelines.

The known immunogenity of RCC has represented the basis supporting theuse of immunotherapy and cancer vaccines in advanced RCC. Theinteresting correlation between lymphocytes PD-1 expression and RCCadvanced stage, grade and prognosis, as well as the selective PD-L1expression by RCC tumor cells and its potential association with worseclinical outcomes, have led to the development of new anti PD-1/PD-L1agents, alone or in combination with anti-angiogenic drugs or otherimmunotherapeutic approaches, for the treatment of RCC (Massari et al.,2015). In advanced RCC, a phase III cancer vaccine trial called TRISTstudy evaluates whether TroVax (a vaccine using a tumor-associatedantigen 5T4, with a pox virus vector), added to first-line standard ofcare therapy, prolongs survival of patients with locally advanced ormRCC. Median survival had not been reached in either group with 399patients (54%) remaining on study however analysis of the data confirmsprior clinical results, demonstrating that TroVax is bothimmunologically active and that there is a correlation between thestrength of the 5T4-specific antibody response and improved survival.Further there are several studies searching for peptide vaccines usingepitopes being over-expressed in RCC.

Various approaches of tumor vaccines have been under investigation.Studies using whole-tumor approaches, including tumor cell lysates,fusions of dendritic cells with tumor cells, or whole-tumor RNA weredone in RCC patients, and remissions of tumor lesions were reported insome of these trials (Avigan et al., 2004; Holtl et al., 2002; Marten etal., 2002; Su et al., 2003; Wittig et al., 2001).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and colorectal cancer, glioblastoma,gastric cancer, hepatocellular carcinoma, and renal cell carcinoma.There is also a need to identify factors representing biomarkers forcancer in general and colorectal cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, and renal cell carcinoma in particular,leading to better diagnosis of cancer, assessment of prognosis, andprediction of treatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.

c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor-(-associated) exon in case of proteins with tumor-specific(-associated) isoforms.

e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis, and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of immense importance for the developmentof pharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell-(CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Longer (elongated) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-I-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way, each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

SUMMARY

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 268 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 268, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 268 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 268,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1P to FIG. 9 all depict embodiments as described herein.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. In Table 1, peptides with SEQ IDNO: 1 to SEQ ID NO: 37 bind to HLA-A*01, peptides with SEQ ID NO: 38 toSEQ ID NO: 61 bind to HLA-A*02, peptides with SEQ ID NO: 62 to SEQ IDNO: 112 bind to HLA-A*03, peptides with SEQ ID NO: 113 to SEQ ID NO: 142bind to HLA-A*24, peptides with SEQ ID NO: 143 to SEQ ID NO: 175 bind toHLA-B*07, peptides with SEQ ID NO: 176 to SEQ ID NO: 194 bind toHLA-B*08, peptides with SEQ ID NO: 195 to SEQ ID NO: 241 bind toHLA-B*44. The peptides in Table 2 have been disclosed before in largelistings as results of high-throughput screenings with high error ratesor calculated using algorithms, but have not been associated with cancerat all before. In Table 2, peptides with SEQ ID NO: 242 to SEQ ID NO:248 bind to HLA-A*01, peptides with SEQ ID NO: 249 to SEQ ID NO: 251bind to HLA-A*02, peptides with SEQ ID NO: 252 to SEQ ID NO: 254 bind toHLA-A*03, peptides with SEQ ID NO: 255 to SEQ ID NO: 259 bind toHLA-B*07, peptides with SEQ ID NO: 260 to SEQ ID NO: 266 bind toHLA-B*44. The peptides in Table 3 are additional peptides that may beuseful in combination with the other peptides of the invention. In Table3, peptide with SEQ ID NO: 267 binds to HLA-A*02, peptide with SEQ IDNO: 268 binds to HLA-A*24.

TABLE 1 Peptides according to the present invention. Seq IDOfficial Gene HLA No Sequence Symbol(s) allotype 1 RSDPVTLDV CEACAM5A*01 2 LPSPTDSNFY EGFR A*01 3 ASSTDSASYY APOB A*01 4 NSDLKYNAL APOB A*015 SILGSDVRVPSY APOB A*01 6 VLDLSTNVY APOB A*01 7 LITGDPKAAYDY COL11A1A*01/A*03 8 TPVTEFSLNTY COL6A3 A*01 9 FITAQNHGY CPS1 A*01 10 ITAQNHGYCPS1 A*01 11 LSAGSGPGQY CPT2 A*01 12 ITFGERFEY CYP2J2 A*01 13 GSTMVEHNYDCBLD2 A*01/A*03 14 YTERDGSAMVY DCLK2 A*01 15 LTDYLKNTY DPP4 A*01 16LSLIDRLVLY EGLN3 A*01 17 YTDKLQHY EPHB2 A*01 18 EVSNGKWLLY ITGA3A*01/A*03 19 VSNGKWLLY ITGA3 A*01/A*03 20 STDEITTRY KLB A*01 21STDIGALMY MMP1 A*01 22 TLEQVQLYY MYO7B A*01 23 TASEDVFQY NOX1 A*01 24YTHHLFIFY NOX1 A*01 25 LMKEVMEHY PLOD2 A*01/B*15 26 EVLDSHIHAY PTPRZ1A*01 27 LDSHIHAY PTPRZ1 A*01 28 LTDYINANY PTPRZ1 A*01 29 SVTDLEMPHYPTPRZ1 A*01/B*18 30 VLDSHIHAY PTPRZ1 A*01 31 VTDLEMPHY PTPRZ1 A*01 32ATVGYFIFY RNF128 A*01 33 FADKIHLAY RNF128 A*01 34 ITDFNNIRYRP11-1220K2.2 A*01 35 FASDLLHLY SLC16A11 A*01 36 YAAYIIHAY TLR3A*01/A*29 37 LTDSFPLKV TTPA A*01 38 VMLNSNVLL AC010879.1, NLGN4X, A*02NLGN4Y 39 YLLPSVVLL AGPAT5 A*02 40 KIDDIWNLEV APOB A*02 41 SLQDTKITLAPOB A*02 42 KMMALVAEL CCDC146 A*02 43 GLMTIVTSL CCL24 A*02 44 SQTGFVVLVCHI3L1 A*02 45 KLLDEVTYL CYP2J2 A*02 46 VLITGLPLI CYP2J2 A*02 47YQDSWFQQL CYP2J2 A*02 48 NLTFIIILI F13B A*02 49 NLASRPYSL F5 A*02 50ELMPRVYTL FAT1 A*02 51 ALAAELNQL GFAP A*02 52 YVSSGEMMV GFAP A*02 53LLMTSLTES LRRN1 A*02 54 YLPPTDPRMSV MMP16 A*02 55 RLWQIQHHL MTCP1 A*0256 FLNQIYTQL MUC5AC A*02 57 GLTGVIMTI NOX1 A*02 58 MLCLLLTL PAEP A*02 59KLHEIYIQA PCDHGC3 A*02 60 GLPDFVKEL RP11-1220K2.2 A*02 61 RLFGLFLNNVTLR3 A*02 62 GSYSALLAKK ABCC2 A*03/A*11 63 KVLGPNGLLK ABCC2 A*03 64STTKLYLAK ABCC2 A*03/A*11 65 VLGPNGLLK ABCC2 A*03/A*68 66 ATYEGIQKKALDH1L1, ALDH1L2 A*03/A*11 67 ATALSLSNK APOB A*03 68 ATAYGSTVSK APOBA*03/A*11 69 ATAYGSTVSKR APOB A*03 70 ATWSASLKNK APOB A*03 71 KLGNNPVSKAPOB A*03 72 KQVFPGLNY APOB A*03 73 KSFDRHFEK APOB A*03/A*11 74QLYSKFLLK APOB A*03 75 QVPTFTIPK APOB A*03 76 SAFGYVFPK APOB A*03/A*1177 SSASLAHMK APOB A*03/A*68 78 STKSTSPPK APOB A*03 79 STNNEGNLK APOBA*03/A*11 80 STSHHLVSR APOB A*03/A*68 81 SVKLQGTSK APOB A*03/A*68 82TAYGSTVSK APOB A*03 83 TAYGSTVSKR APOB A*03/A*68 84 TVASLHTEK APOBA*03/A*68 85 KMAAWPFSR C4BPA A*03 86 KTPSGALHRK C4BPA A*03/A*11 87SSYSRSSAVK DCLK2 A*03 88 MLLQQPLIY DNAH11 A*03 89 KITDFGLAK EGFR A*03 90GSRLGKYYVK EGLN3 A*03 91 SLIDRLVLY EGLN3 A*03 92 AVLDLGSLLAK FAM149AA*03 93 ALDKPGKSK FAM181B A*03 94 KTYVGHPVKM FAT1 A*03 95 RLFESSFHYGAL3ST1 A*03/A*29 96 FSLAGALNAGFK GFAP A*03 97 RMPPPLPTR GFAP A*03 98KLYPTYSTK GPLD1 A*03 99 ATMQSKLIQK GRM8 A*03 100 ALLGVIIAK HAVCR1 A*03101 GVIIAKKYFFK HAVCR1 A*03/A*11 102 IIAKKYFFK HAVCR1 A*03 103 KSWTASSSYLOXL2 A*03 104 STQDTLLIK MXRA5 A*03 105 GSAALYLLR NDUFA4L2 A*03 106RLSPNDQYK NDUFA4L2 A*03 107 EIYGGHHAGF OLIG2 A*03 108 LLKSSVGNFY PCDHB8A*03 109 KIIAPLVTR PLOD2 A*03/A*11 110 GTESGTILK SEMA5B A*03/A*11 111KIKEHVRSK UBD A*03 112 KMMADYGIRK UBD A*03 113 VWAKILSAF ABCB4 A*24 114KFLDSNIKF APOB A*24/A*23 115 YFEEAANFL BAAT A*24 116 LVLDYSKDYNHW CPS1A*24 117 NFLPPIIARF DCBLD2 A*24 118 TYISKTIAL EXOC3L2 A*24 119 YMKALGVGFFABP7 A*24/B*15 120 MYAKEFDLL FMO5 A*24 121 SYIEKVRFL GFAP A*24 122KLYGMPTDFGF GRB7 A*24/A*32 123 RQYLAINQI ITPR2 A*24 124 EVYSPEADQWKLHDC8A A*24/A*25 125 IYGPKYIHPSF MACC1 A*24/A*23 126 TFQDKTLNF MACC1A*24 127 IFINLSPEF MUC5AC A*24 128 SYTKVEARL MUC5AC A*24 129 VFLNQIYTQLMUC5AC A*24 130 VYGDGHYLTF MUC5AC A*24 131 KQLDHNLTF NOX1 A*24/B*15 132VYNPVIYVF OPN3 A*24 133 SFDSNLLSF PIWIL1 A*24 134 TYLTGRQF PLCB4 A*24135 VIAPIISNF SLC12A2 A*24/B*15 136 EYNNIQHLF TLR3 A*24 137 KYLSLSNSFTLR3 A*24 138 KYLSIPTVF UGT1A3 A*24 139 PYASLASELFUGT1A3, UGT1A4, UGT1A5 A*24 140 KYLSIPAVF UGT1A4, UGT1A5 A*24 141KYLSIPAVFF UGT1A4, UGT1A5 A*24 142 SSFPGAGNTW WSCD1 A*24/A*25 143FELPTGAGLQL APOB B*07 144 IPEPSAQQL APOB B*07 145 RVPSYTLIL APOB B*07146 SPGDKRLAA APOB B*07 147 SPIKVPLLL APOB B*07 148 VPDGVSKVL APOB B*07149 YPLTGDTRL APOB B*07 150 KPSSKALGTSL ATP10B B*07 151 VVHPRTLLL CYP2J2B*07/B*15 152 IPSRLLAIL EFNA5 B*07 153 APAAVPSAPA FEZF1 B*07 154GPGTRLSL GFAP B*07 155 FPYPYAERL GRIN2D B*07/B*35 156 HPQVVILSL HAVCR1B*07/B*35 157 SPSPGKDPTL HSF4 B*07 158 VPERGEPEL HSF4 B*07 159 FPAHPSLLLITGA3 B*07 160 RPAPADSAL KISS1R B*07 161 NPYEGRVEV LOXL2 B*07/B*51 162MPMISIPRV LPPR5 B*07/B*51 163 RPASSLRP MMP11 B*07 164 ISTPSEVSTPL MUC17B*07 165 TPIAKVSEL NKD1 B*07 166 HDPDVGSNSL PCDHGC3 B*07 167 YPSEVEHMFPGF B*07/B*35 168 IPTDKLLVI PLOD2 B*07 169 FPTEVTPHAF PTPRZ1 B*07 170SPMWHVQQL QRFPR B*07 171 APKLFAVAF SEC14L6 B*07 172 KPAHYPLIAL TEX11B*07 173 MVPSAGQLALF TGFA B*07 174 VPSLQRLML TLR3 B*07 175 HPIETLVDIFVEGFA B*07/B*35 176 AAMSRYEL APOB B*08 177 DLKYNALDL APOB B*08 178HAKEKLTAL APOB B*08 179 IQIYKKLRTSSF APOB B*08 180 LLKAEPLAF APOBB*08/B*15 181 YKKLRTSSF APOB B*08 182 LPFLRENDL ASTN1 B*08/B*07 183FQKLKLLSL ATP10B B*08 184 EPVKKSRL CCND1 B*08 185 NPNLKTLL CHI3L1 B*08186 SLIDRLVL EGLN3 B*08/B*07 187 YVKERSKAM EGLN3 B*08 188 SALDHVTRLEXOC3L2 B*08 189 HIFLRTTL ITPR2 B*08 190 SRSMRLLLL REG4 B*08 191LINLKYLSL TLR3 B*08 192 LPMLKVLNL TLR3 B*08 193 LSYNKYLQL TLR3 B*08 194EAKRHLLQV UBD B*08 195 AEAVLKTLQEL APOB B*44/B*40 196 AEQTGTWKL APOBB*44 197 EEAKQVLFL APOB B*44 198 FELPTGAGL APOB B*44/B*40 199 GEATLQRIYAPOB B*44 200 GEELGFASL APOB B*44 201 GEHTSKATL APOB B*44 202 KEFNLQNMGLAPOB B*44 203 KENFAGEATL APOB B*44 204 KESQLPTVM APOB B*44 205 QEVLLQTFLAPOB B*44 206 SEPINIIDAL APOB B*44/B*40 207 TEATMTFKY APOB B*44 208AEHDAVRNAL ASCL2 B*44 209 YEVDTVLRY BCAN B*44 210 SENIVIQVY C5 B*44 211TEKEMIQKL CCDC146 B*44 212 AEETCAPSV CCND1 B*44/B*51 213 TTMDQKSLWCHI3L2 B*44 214 AEQPDGLIL CPS1 B*44 215 AFITAQNHGY CPS1 B*44 216LQEEKVPAIY CPS1 B*44 217 NEINEKIAPSF CPS1 B*44 218 AEGGKVPIKW EGFR B*44219 AENAEYLRV EGFR B*44 220 KEITGFLLI EGFR B*44 221 AEERAEAKKKF EGLN3B*44 222 NEISTFHNL GPC3 B*44 223 SEVPVARVW IGFBP1 B*44 224 SESAVFHGFITGA3 B*44 225 SEAFPSRAL KISS1R B*44 226 EELLHGQLF MUC5AC B*44 227TEHTQSQAAW NXPH4 B*44 228 AEKQTPDGRKY PCDHGB2 B*44 229 KESDGFHRF PLOD2B*44 230 AENLFRAFL PRKDC B*44 231 AEIHTAEI PTHLH B*44 232 AEKDGKLTDYPTPRZ1 B*44 233 DESEKTTKSF PTPRZ1 B*44 234 EEESLLTSF PTPRZ1 B*44 235EEFETLKEF PTPRZ1 B*44 236 EEKLIIQDF PTPRZ1 B*44 237 LEMPHYSTF PTPRZ1B*44 238 SENPETITY PTPRZ1 B*44 239 TEVLDSHIHAY PTPRZ1 B*44 240 HELENHSMYTRIM9 B*44 241 REAEPIPKM TRIO B*44

TABLE 2 Additional peptides according to the presentinvention with no prior known cancer association. Seq ID Official GeneHLA No Sequence Symbol(s) allotype 242 FSDKELAAY ABCB4 A*01 243RSPNNFLSY CCND1 A*01/A*03 244 RSDPVTLNV CEACAM1, CEACAM6, A*01CEACAM7, PSG1, PSG4, PSG5, PSG7 245 ITEKNSGLY CEACAM5 A*01 246 YSDLHAFYYMANEAL A*01 247 RSDPGGGGLAY MEX3B A*01 248 YSHAAGQGTGLY SOX9 A*01 249ALFPERITV ATAT1 A*02 250 KMILKMVQL FRAME A*02 251 RLASRPLLL PTGFRN A*02252 RIYNGIGVSR DCBLD2 A*03 253 KLFGTSGQK EGFR A*03 254 AVATKFVNK TRIOA*03 255 LPDGSRVEL ACTL8 B*07 256 LPALPQQLI COL6A3 B*07 257 SPLRGGSSLEFNA3, EFNA4 B*07 258 APSGTRVVQVL PCDHGC3 B*07 259 RPAVGHSGL ZC3H3 B*07260 EEAPLVTKAF ASPSCR1 B*44 261 IEALLESSL CCND1 B*44 262 MELLLVNKL CCND1B*44 263 QQATPGPAY CEA, CEACAM5, B*44 CEACAM6 264 DEYLIPQQGF EGFR B*44265 EEVDVPIKLY EPHB1, EPHB2 B*44 266 ARLTPIPFGL TMEM64 B*44

TABLE 3 Peptides of the invention useful for e.g.personalized cancer therapies Seq ID HLA No SequenceOfficial Gene Symbol(s) allotype 267 KTLGKLWRL SOX10, SOX8, SOX9 A*02268 DYIPYVFKL APOB A*24

The present invention generally relates to the peptides according to thepresent invention for use in the treatment of proliferative diseases,such as, for example, chronic lymphocytic leukemia, chronic myeloidleukemia and acute myeloid leukemia, and other lymphoid neoplasms, forexample, Non-Hodgkin lymphoma, post-transplant lymphoproliferativedisorders (PTLD) as well as other myeloid neoplasms, such as primarymyelofibrosis, essential thrombocytopenia, polycythemia vera, as well asother neoplasms such as esophageal cancer, non-small cell lung cancer,small cell lung cancer, pancreatic cancer, prostate cancer, melanoma,breast cancer, gallbladder cancer and cholangiocarcinoma, urinarybladder cancer, uterine cancer, head and neck squamous cell carcinoma,mesothelioma.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 268. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 241 (see Table 1), and their uses in theimmunotherapy of colorectal cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, and renal cell carcinoma, chronic lymphocyticleukemia, chronic myeloid leukemia and acute myeloid leukemia, and otherlymphoid neoplasms, for example, Non-Hodgkin lymphoma, post-transplantlymphoproliferative disorders (PTLD) as well as other myeloid neoplasms,such as primary myelofibrosis, essential thrombocytopenia, polycythemiavera, as well as other neoplasms such as esophageal cancer, non-smallcell lung cancer, small cell lung cancer, pancreatic cancer, prostatecancer, melanoma, breast cancer, gallbladder cancer andcholangiocarcinoma, urinary bladder cancer, uterine cancer, head andneck squamous cell carcinoma, mesothelioma, and preferably colorectalcancer, glioblastoma, gastric cancer, hepatocellular carcinoma, andrenal cell carcinoma.

Another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofcolorectal cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, and renal cell carcinoma, chronic lymphocytic leukemia,chronic myeloid leukemia and acute myeloid leukemia, and other lymphoidneoplasms, for example, Non-Hodgkin lymphoma, post-transplantlymphoproliferative disorders (PTLD) as well as other myeloid neoplasms,such as primary myelofibrosis, essential thrombocytopenia, polycythemiavera, as well as other neoplasms such as esophageal cancer, non-smallcell lung cancer, small cell lung cancer, pancreatic cancer, prostatecancer, melanoma, breast cancer, gallbladder cancer andcholangiocarcinoma, urinary bladder cancer, uterine cancer, head andneck squamous cell carcinoma, mesothelioma.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 268.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 268, preferably containing SEQ IDNo. 1 to SEQ ID No. 241, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are colorectal cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, and renal cellcarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia andacute myeloid leukemia, and other lymphoid neoplasms, for example,Non-Hodgkin lymphoma, post-transplant lymphoproliferative disorders(PTLD) as well as other myeloid neoplasms, such as primarymyelofibrosis, essential thrombocytopenia, polycythemia vera, as well asother neoplasms such as esophageal cancer, non-small cell lung cancer,small cell lung cancer, pancreatic cancer, prostate cancer, melanoma,breast cancer, gallbladder cancer and cholangiocarcinoma, urinarybladder cancer, uterine cancer, head and neck squamous cell carcinoma,mesothelioma, and preferably colorectal cancer, glioblastoma, gastriccancer, hepatocellular carcinoma, and renal cell carcinoma cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably colorectalcancer, glioblastoma, gastric cancer, hepatocellular carcinoma, andrenal cell carcinoma. The marker can be over-presentation of thepeptide(s) themselves, or over-expression of the corresponding gene(s).The markers may also be used to predict the probability of success of atreatment, preferably an immunotherapy, and most preferred animmunotherapy targeting the same target that is identified by thebiomarker. For example, an antibody or soluble TCR can be used to stainsections of the tumor to detect the presence of a peptide of interest incomplex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, or 12 or longer, and incase of MHC class II peptides (elongated variants of the peptides of theinvention) they can be as long as 13, 14, 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. Preferably,the salts are pharmaceutical acceptable salts of the peptides, such as,for example, the chloride or acetate (trifluoroacetate) salts. It has tobe noted that the salts of the peptides according to the presentinvention differ substantially from the peptides in their state(s) invivo, as the peptides are not salts in vivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the oligopeptide is not critical to the invention, as long as thecorrect epitope or epitopes are maintained therein. The oligopeptidesare typically less than about 30 amino acid residues in length, andgreater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans, there are three different genetic loci that encode MHC classI molecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 4 Expression frequencies F of HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44 serotypes. Haplotype frequenciesGf are derived from a study which used HLA-typing data from a registryof more than 6.5 million volunteer donors in the U.S. (Gragert et al.,2013). The haplotype frequency is the frequency of a distinct allele onan individual chromosome. Due to the diploid set of chromosomes withinmammalian cells, the frequency of genotypic occurrence of this allele ishigher and can be calculated employing the Hardy-Weinberg principle (F =1 − (1-Gf)²). Calculated phenotype from allele Allele Populationfrequency (F) A*02 African (N = 28557) 32.3% European Caucasian (N =1242890) 49.3% Japanese (N = 24582) 42.7% Hispanic, S + Cent Amer. 46.1%(N = 146714) Southeast Asian (N = 27978) 30.4% A*01 African (N = 28557)10.2% European Caucasian (N = 1242890) 30.2% Japanese (N = 24582)  1.8%Hispanic, S + Cent Amer. 14.0% (N = 146714) Southeast Asian (N = 27978)21.0% A*03 African (N = 28557) 14.8% European Caucasian (N = 1242890)26.4% Japanese (N = 24582)  1.8% Hispanic, S + Cent Amer. 14.4% (N =146714) Southeast Asian (N =27978) 10.6% A*24 African (N = 28557)  2.0%European Caucasian (N = 1242890)  8.6% Japanese (N = 24582) 35.5%Hispanic, S + Cent Amer. 13.6% (N = 146714) Southeast Asian (N = 27978)16.9% B*07 African (N = 28557) 14.7% European Caucasian (N = 1242890)25.0% Japanese (N = 24582) 11.4% Hispanic, S + Cent Amer. 12.2% (N =146714) Southeast Asian (N = 27978) 10.4% B*08 African (N = 28557)  6.0%European Caucasian (N = 1242890) 21.6% Japanese (N = 24582)  1.0%Hispanic, S + Cent Amer.  7.6% (N = 146714) Southeast Asian (N = 27978) 6.2% B*44 African (N = 28557) 10.6% European Caucasian (N = 1242890)26.9% Japanese (N = 24582) 13.0% Hispanic, S + Cent Amer. 18.2% (N =146714) Southeast Asian (N = 27978) 13.1%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*02, A*01, A*03, A*24,B*07, B*08 or B*44. A vaccine may also include pan-binding MHC class IIpeptides. Therefore, the vaccine of the invention can be used to treatcancer in patients that are A*02-, A*01-, A*03-, A*24-, B*07-, B*08- orB*44-positive, whereas no selection for MHC class II allotypes isnecessary due to the pan-binding nature of these peptides.

If A*02 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86%.

TABLE 5 HLA alleles coverage in European Caucasian population(calculated from (Gragert et al., 2013)). coverage (at least combinedone A- combined combined with B*07 allele) with B*07 with B*44 and B*44A*02/A*01 70% 78% 78% 84% A*02/A*03 68% 76% 76% 83% A*02/A*24 61% 71%71% 80% A*′01/A*03 52% 64% 65% 75% A*01/A*24 44% 58% 59% 71% A*03/A*2440% 55% 56% 69% A*02/A*01/A*03 84% 88% 88% 91% A*02/A*01/A*24 79% 84%84% 89% A*03/A*02/A*24 77% 82% 83% 88% A*03/A*01/A*24 63% 72% 73% 81%A*02/A*01/A*03/A*24 90% 92% 93% 95%

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:percent identity=100[1−(C/R)]wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and

(iiii) the alignment has to start at position 1 of the alignedsequences;

and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 268 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 268, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 268. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way, it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 268, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acid whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptidesaccording to SEQ ID NO: 1, 21, 48, 250, 91, 253, 117, 125, 160, 255,178, 183, 229, and 232. Position 1 2 3 4 5 6 7 8 9 SEQ ID NO: 1 R S D PV T L D V Variant Y SEQ ID NO: 272 SEQ ID NO: 273 A SEQ ID NO: 274 E YSEQ ID NO: 275 E A SEQ ID NO: 276 T Y SEQ ID NO: 277 T A SEQ ID NO: 278T E Y SEQ ID NO: 279 T E A Position 1 2 3 4 5 6 7 8 9 SEQ. ID NO: 21 S TD I G A L M Y Variant S SEQ ID NO: 280 SEQ ID NO: 281 S A SEQ ID NO: 282S E SEQ ID NO: 283 S E A SEQ ID NO: 284 A SEQ ID NO: 285 ESEQ ID NO: 286 E A Position 1 2 3 4 5 6 7 8 9 SEQ. ID NO: 48 N L T F I II L I Variant V SEQ ID NO: 287 SEQ ID NO: 288 L SEQ ID NO: 289 ASEQ ID NO: 290 M V SEQ ID NO: 291 M SEQ ID NO: 292 M L SEQ ID NO: 293 MA SEQ ID NO: 294 A V SEQ ID NO: 295 A SEQ ID NO: 296 A L SEQ ID NO: 297A A SEQ ID NO: 298 V V SEQ ID NO: 299 V SEQ ID NO: 300 V LSEQ ID NO: 301 V A SEQ ID NO: 302 T V SEQ ID NO: 303 T SEQ ID NO: 304 TL SEQ ID NO: 305 T A SEQ ID NO: 306 Q V SEQ ID NO: 307 Q SEQ ID NO: 308Q L SEQ ID NO: 309 Q A Position 1 2 3 4 5 6 7 8 9 SEQ ID NO: 250 K M I LK M V Q L Variant L V SEQ ID NO: 310 SEQ ID NO: 311 L I SEQ ID NO: 312 LSEQ ID NO: 313 L A SEQ ID NO: 314 V SEQ ID NO: 315 I SEQ ID NO: 316 ASEQ ID NO: 317 A V SEQ ID NO: 318 A I SEQ ID NO: 319 A SEQ ID NO: 320 AA SEQ ID NO: 321 V V SEQ ID NO: 322 V I SEQ ID NO: 323 V SEQ ID NO: 324V A SEQ ID NO: 325 T V SEQ ID NO: 326 T I SEQ ID NO: 327 TSEQ ID NO: 328 T A SEQ ID NO: 329 Q V SEQ ID NO: 330 Q I SEQ ID NO: 331Q SEQ ID NO: 332 Q A Position 1 2 3 4 5 6 7 8 9 SEQ ID NO: 91 S L I D RL V L Y Variant K SEQ ID NO: 333 SEQ ID NO: 334 R SEQ ID NO: 335 FSEQ ID NO: 336 I K SEQ ID NO: 337 I SEQ ID NO: 338 I R SEQ ID NO: 339 IF SEQ ID NO: 340 M K SEQ ID NO: 341 M SEQ ID NO: 342 M R SEQ ID NO: 343M F SEQ ID NO: 344 V K SEQ ID NO: 345 V SEQ ID NO: 346 V RSEQ ID NO: 347 V F SEQ ID NO: 348 T K SEQ ID NO: 349 T SEQ ID NO: 350 TR SEQ ID NO: 351 T F Position 1 2 3 4 5 6 7 8 9 SEQ ID NO: 253 K L F G TS G Q K Variant Y SEQ ID NO: 352 SEQ ID NO: 353 R SEQ ID NO: 354 FSEQ ID NO: 355 I SEQ ID NO: 356 I Y SEQ ID NO: 357 I R SEQ ID NO: 358 IF SEQ ID NO: 359 M SEQ ID NO: 360 M Y SEQ ID NO: 361 M R SEQ ID NO: 362M F SEQ ID NO: 363 V SEQ ID NO: 364 V Y SEQ ID NO: 365 V RSEQ ID NO: 366 V F SEQ ID NO: 367 T SEQ ID NO: 368 T Y SEQ ID NO: 369 TR SEQ ID NO: 370 T F Position 1 2 3 4 5 6 7 8 9 10 SEQ ID NO: 117 N F LP P I I A R F Variant Y I SEQ ID NO: 371 SEQ ID NO: 372 Y LSEQ ID NO: 373 Y SEQ ID NO: 374 I SEQ ID NO: 375 L Position 1 2 3 4 5 67 8 9 10 11 SEQ ID NO: 125 I Y G P K Y I H P S F Variant ISEQ ID NO: 376 SEQ ID NO: 377 L SEQ ID NO: 378 F I SEQ ID NO: 379 F LSEQ ID NO: 380 F Position 1 2 3 4 5 6 7 8 9 SEQ ID NO: 160 R P A P A D SA L Variant F SEQ ID NO: 381 SEQ ID NO: 382 V SEQ ID NO: 383 MSEQ ID NO: 384 A SEQ ID NO: 385 I Position 1 2 3 4 5 6 7 8 9SEQ ID NO: 255 L P D G S R V E L Variant F SEQ ID NO: 386 SEQ ID NO: 387V SEQ ID NO: 388 M SEQ ID NO: 389 A SEQ ID NO: 390 I Position 1 2 3 4 56 7 8 9 SEQ ID NO: 178 H A K E K L T A L Variant V SEQ ID NO: 391SEQ ID NO: 392 I SEQ ID NO: 393 M SEQ ID NO: 394 F SEQ ID NO: 395 RSEQ ID NO: 396 R V SEQ ID NO: 397 R I SEQ ID NO: 398 R M SEQ ID NO: 399R F SEQ ID NO: 400 H SEQ ID NO: 401 H V SEQ ID NO: 402 H ISEQ ID NO: 403 H M SEQ ID NO: 404 H F SEQ ID NO: 405 R SEQ ID NO: 406 RV SEQ ID NO: 407 R I SEQ ID NO: 408 R M SEQ ID NO: 409 R FSEQ ID NO: 410 R R SEQ ID NO: 411 R R V SEQ ID NO: 412 R R ISEQ ID NO: 413 R R M SEQ ID NO: 414 R R F SEQ ID NO: 415 R HSEQ ID NO: 416 R H V SEQ ID NO: 417 R H I SEQ ID NO: 418 R H MSEQ ID NO: 419 R H F SEQ ID NO: 420 L SEQ ID NO: 421 L V SEQ ID NO: 422L I SEQ ID NO: 423 L M SEQ ID NO: 424 L F SEQ ID NO: 425 L RSEQ ID NO: 426 L R V SEQ ID NO: 427 L R I SEQ ID NO: 428 L R MSEQ ID NO: 429 L R F SEQ ID NO: 430 L H SEQ ID NO: 431 L H VSEQ ID NO: 432 L H I SEQ ID NO: 433 L H M SEQ ID NO: 434 L H F Position1 2 3 4 5 6 7 8 9 SEQ ID NO: 183 F Q K L K L L S L Variant VSEQ ID NO: 435 SEQ ID NO: 436 I SEQ ID NO: 437 M SEQ ID NO: 438 FSEQ ID NO: 439 R SEQ ID NO: 440 R V SEQ ID NO: 441 R I SEQ ID NO: 442 RM SEQ ID NO: 443 R F SEQ ID NO: 444 H SEQ ID NO: 445 H V SEQ ID NO: 446H I SEQ ID NO: 447 H M SEQ ID NO: 448 H F SEQ ID NO: 449 RSEQ ID NO: 450 R V SEQ ID NO: 451 R I SEQ ID NO: 452 R M SEQ ID NO: 453R F SEQ ID NO: 454 R R SEQ ID NO: 455 R R V SEQ ID NO: 456 R R ISEQ ID NO: 457 R R M SEQ ID NO: 458 R R F SEQ ID NO: 459 R HSEQ ID NO: 460 R H V SEQ ID NO: 461 R H I SEQ ID NO: 462 R H MSEQ ID NO: 463 R H F SEQ ID NO: 464 L SEQ ID NO: 465 L V SEQ ID NO: 466L I SEQ ID NO: 467 L M SEQ ID NO: 468 L F SEQ ID NO: 469 L RSEQ ID NO: 470 L R V SEQ ID NO: 471 L R I SEQ ID NO: 472 L R MSEQ ID NO: 473 L R F SEQ ID NO: 474 L H SEQ ID NO: 475 L H VSEQ ID NO: 476 L H I SEQ ID NO: 477 L H M SEQ ID NO: 478 L H F Position1 2 3 4 5 6 7 8 9 SEQ ID NO: 229 K E S D G F H R F Variant WSEQ ID NO: 479 SEQ ID NO: 480 Y SEQ ID NO: 481 L SEQ ID NO: 482 DSEQ ID NO: 483 D W SEQ ID NO: 484 D Y SEQ ID NO: 485 D L Position 1 2 34 5 6 7 8 9 10 SEQ ID NO: 232 A E K D G K L T D Y Variant FSEQ ID NO: 486 SEQ ID NO: 487 W SEQ ID NO: 488 L SEQ ID NO: 489 D FSEQ ID NO: 490 D W SEQ ID NO: 491 D SEQ ID NO: 492 D L

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus C-terminus N-terminus 4 0 3 0or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, if they have substantially identical antigenicactivity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 268.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 268 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond, amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

-   -   Briefly, modification of e.g. arginyl residues in proteins is        often based on the reaction of vicinal dicarbonyl compounds such        as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to        form an adduct. Another example is the reaction of methylglyoxal        with arginine residues. Cysteine can be modified without        concomitant modification of other nucleophilic sites such as        lysine and histidine. As a result, a large number of reagents        are available for the modification of cysteine. The websites of        companies such as Sigma-Aldrich provide information on specific        reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetyl imidazole can be used for themodification of tyrosyl residues. Cross-linking via the formation ofdityrosine can be accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 268and has been synthetically produced (e.g. synthesized) as apharmaceutically acceptable salt. Methods to synthetically producepeptides are well known in the art. The salts of the peptides accordingto the present invention differ substantially from the peptides in theirstate(s) in vivo, as the peptides as generated in vivo are no salts. Thenon-natural salt form of the peptide mediates the solubility of thepeptide, in particular in the context of pharmaceutical compositionscomprising the peptides, e.g. the peptide vaccines as disclosed herein.A sufficient and at least substantial solubility of the peptide(s) isrequired in order to efficiently provide the peptides to the subject tobe treated. Preferably, the salts are pharmaceutically acceptable saltsof the peptides. These salts according to the invention include alkalineand earth alkaline salts such as salts of the Hofmeister seriescomprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻,I⁻, SCN⁻ and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺,Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄,(NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃,NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO,Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄,K₂SO₄, KCH₃COO, KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄,NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄,CsI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr,LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂,Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂,MnCl₂, Ca₃(PO₄), Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂, CaBr₂,Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂,BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, andBa(SCN)₂. Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄,KCl, NaCl, and CaCl₂, such as, for example, the chloride or acetate(trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine inN,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethyl acrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoracetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoracetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESl) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from colorectal cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, and renal cellcarcinoma samples (N=35 CRC, 12 GBM, 10 GC, 22 HCC, and 79 RCC samples)with the fragmentation patterns of corresponding synthetic referencepeptides of identical sequences. Since the peptides were directlyidentified as ligands of HLA molecules of primary tumors, these resultsprovide direct evidence for the natural processing and presentation ofthe identified peptides on primary cancer tissue obtained from 158colorectal cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, and renal cell carcinoma patients (cf. Example 1).

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

HLA-peptide complexes from colorectal cancer, glioblastoma, gastriccancer, hepatocellular carcinoma, and renal cell carcinoma tissuesamples were purified and HLA-associated peptides were isolated andanalyzed by LC-MS (see example 1). All TUMAPs contained in the presentapplication were identified with this approach on primary colorectalcancer, glioblastoma, gastric cancer, hepatocellular carcinoma, andrenal cell carcinoma samples confirming their presentation on primarycolorectal cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, and renal cell carcinoma.

Besides presentation of the peptide, mRNA expression of the underlyinggene was tested. mRNA data were obtained via RNASeq analyses of normaltissues and cancer tissues (cf. Example 2, FIG. 1). Peptides which arederived from proteins whose coding mRNA is highly expressed in cancertissue, but very low or absent in vital normal tissues, were preferablyincluded in the present invention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably colorectal cancer, glioblastoma, gastriccancer, hepatocellular carcinoma, and renal cell carcinoma that over- orexclusively present the peptides of the invention. These peptides wereshown by mass spectrometry to be naturally presented by HLA molecules onprimary human colorectal cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, and renal cell carcinoma samples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy brain cells, kidney cells, liver cells, large intestine cells,stomach cells or other normal tissue cells, demonstrating a high degreeof tumor association of the source genes (see Example 2). Moreover, thepeptides themselves are presented on tumor tissue—“tumor tissue” inrelation to this invention shall mean a sample from a patient sufferingfrom colorectal cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, and renal cell carcinoma.

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. colorectal cancer, glioblastoma, gastriccancer, hepatocellular carcinoma, and renal cell carcinoma cellspresenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well (see also below). Thus, the peptides of the presentinvention are useful for generating an immune response in a patient bywhich tumor cells can be destroyed. An immune response in a patient canbe induced by direct administration of the described peptides orsuitable precursor substances (e.g. elongated peptides, proteins, ornucleic acids encoding these peptides) to the patient, ideally incombination with an agent enhancing the immunogenicity (i.e. anadjuvant). The immune response originating from such a therapeuticvaccination can be expected to be highly specific against tumor cellsbecause the target peptides of the present invention are not presentedon normal tissues in comparable copy numbers, preventing the risk ofundesired autoimmune reactions against normal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides according to the invention capable of binding toTCRs and antibodies when presented by an MHC molecule.

The present description also relates to fragments of the TCRs accordingto the invention that are capable of binding to a peptide antigenaccording to the present invention when presented by an HLA molecule.The term particularly relates to soluble TCR fragments, for example TCRsmissing the transmembrane parts and/or constant regions, single chainTCRs, and fusions thereof to, for example, with Ig.

The present description also relates to nucleic acids, vectors and hostcells for expressing TCRs and peptides of the present description; andmethods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment, the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise, the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to a peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta heterodimeric TCRs of the present description may have a TRACconstant domain sequence and a TRBC1 or TRBC2 constant domain sequence,and the TRAC constant domain sequence and the TRBC1 or TRBC2 constantdomain sequence of the TCR may be linked by the native disulfide bondbetween Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a peptide-HLA molecule complex, which is at leastdouble that of a TCR comprising the unmutated TCR alpha chain and/orunmutated TCR beta chain. Affinity-enhancement of tumor-specific TCRs,and its exploitation, relies on the existence of a window for optimalTCR affinities. The existence of such a window is based on observationsthat TCRs specific for HLA-A2-restricted pathogens have KD values thatare generally about 10-fold lower when compared to TCRs specific forHLA-A2-restricted tumor-associated self-antigens. It is now known,although tumor antigens have the potential to be immunogenic, becausetumors arise from the individual's own cells only mutated proteins orproteins with altered translational processing will be seen as foreignby the immune system. Antigens that are upregulated or overexpressed (socalled self-antigens) will not necessarily induce a functional immuneresponse against the tumor: T-cells expressing TCRs that are highlyreactive to these antigens will have been negatively selected within thethymus in a process known as central tolerance, meaning that onlyT-cells with low-affinity TCRs for self-antigens remain. Therefore,affinity of TCRs or variants of the present description to peptides canbe enhanced by methods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with a peptide, incubating PBMCs obtained from the transgenic micewith tetramer-phycoerythrin (PE), and isolating the high avidity T-cellsby fluorescence activated cell sorting (FACS)-Calibur analysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced (Schmitt et al., 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “op-timal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion) (Schmitt et al., 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments, the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments, theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluene sulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient, or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pure,or combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 268, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Yeps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG® (FLAGepitope), 3xFLAG® (FLAG epitope), c-myc or MAT. These fusion proteinsallow for detection, purification and analysis of recombinant protein.Dual-tagged fusions provide flexibility in detection.

-   -   The strong human cytomegalovirus (CMV) promoter regulatory        region drives constitutive protein expression levels as high as        1 mg/L in COS cells. For less potent cell lines, protein levels        are typically −0.1 mg/L. The presence of the SV40 replication        origin will result in high levels of DNA replication in SV40        replication permissive COS cells. CMV vectors, for example, can        contain the pMB1 (derivative of pBR322) origin for replication        in bacterial cells, the b-lactamase gene for ampicillin        resistance selection in bacteria, hGH poly A, and the f1 origin.        Vectors containing the pre-pro-trypsin leader (PPT) sequence can        direct the secretion of FLAG® (FLAG epitope) fusion proteins        into the culture medium for purification using ANTI-FLAG® (FLAG        epitope) antibodies, resins, and plates. Other vectors and        expression systems are well known in the art for use with a        variety of host cells.    -   In another embodiment two or more peptides or peptide variants        of the invention are encoded and thus expressed in a successive        order (similar to “beads on a string” constructs). In doing so,        the peptides or peptide variants may be linked or fused together        by stretches of linker amino acids, such as for example LLLLLL        (SEQ ID NO: 271), or may be linked without any additional        peptide(s) between them. These constructs can also be used for        cancer therapy, and may induce immune responses both involving        MHC I and MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment, the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment, the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CDS-positive T cellsand helper-T (TH) cells to an antigen, and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FL T3 ligand, GM-CSF, 1030, 1031, Imiquimod(ALDARA®), resiquimod, IMUFACT®, IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, JUVIMMUNE®, LIPOVAC®, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK®, OspA, PEPTEL® vector system,poly(lactid coglycolid) [PLG]-based and dextran microparticles,talactoferrin SRL 172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,QUIL®, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also, cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivatives thereof (e.g., AMPLIGEN® (rintatolimod),HILTONOL® (poly-ICLC), poly-(ICLC), poly(IC-R), poly(I:C12U), non-CpGbacterial DNA or RNA as well as immunoactive small molecules andantibodies such as cyclophosphamide, sunitinib, bevacizumab, CELEBREX®(celecoxib), NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib,temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap,ZD2171, AZD2171, anti-CTLA4, other antibodies targeting key structuresof the immune system (e.g. anti-CD40, anti-TGFbeta, anti-TNFalphareceptor) and SC58175, which may act therapeutically and/or as anadjuvant. The amounts and concentrations of adjuvants and additivesuseful in the context of the present invention can readily be determinedby the skilled artisan without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore, differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment, a scaffold is ableto activate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting another peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labeling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example, afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 268,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen (preferably a peptide according to thepresent invention), the method comprising: immunizing a geneticallyengineered non-human mammal comprising cells expressing said human majorhistocompatibility complex (MHC) class I or II with a soluble form of aMHC class I or II molecule being complexed with said HLA-restrictedantigen; isolating mRNA molecules from antibody producing cells of saidnon-human mammal; producing a phage display library displaying proteinmolecules encoded by said mRNA molecules; and isolating at least onephage from said phage display library, said at least one phagedisplaying said antibody specifically binding to said human majorhistocompatibility complex (MHC) class I or II being complexed with saidHLA-restricted antigen.

It is thus a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with a HLA-restricted antigen, wherein theantibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 268, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 268 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:268 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 268, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 268.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of colorectal cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, and renal cellcarcinoma.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 268 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are colorectal cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, and renal cellcarcinoma cells or other solid or hematological tumor cells such aschronic lymphocytic leukemia, chronic myeloid leukemia and acute myeloidleukemia, and other lymphoid neoplasms, for example, Non-Hodgkinlymphoma, post-transplant lymphoproliferative disorders (PTLD) as wellas other myeloid neoplasms, such as primary myelofibrosis, essentialthrombocytopenia, polycythemia vera, as well as other neoplasms such asesophageal cancer, non-small cell lung cancer, small cell lung cancer,pancreatic cancer, prostate cancer, melanoma, breast cancer, gallbladdercancer and cholangiocarcinoma, urinary bladder cancer, uterine cancer,head and neck squamous cell carcinoma, mesothelioma.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of colorectal cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, and renal cell carcinoma. The presentinvention also relates to the use of these novel targets for cancertreatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of a colorectal cancer, glioblastoma,gastric cancer, hepatocellular carcinoma, and renal cell carcinomamarker (poly)peptide, delivery of a toxin to a colorectal cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, and renal cellcarcinoma cell expressing a cancer marker gene at an increased level,and/or inhibiting the activity of a colorectal cancer, glioblastoma,gastric cancer, hepatocellular carcinoma, and renal cell carcinomamarker polypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length colorectal cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, and renal cell carcinoma marker polypeptidesor fragments thereof may be used to generate the antibodies of theinvention. A polypeptide to be used for generating an antibody of theinvention may be partially or fully purified from a natural source, ormay be produced using recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 268polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the colorectal cancer, glioblastoma,gastric cancer, hepatocellular carcinoma, and renal cell carcinomamarker polypeptide used to generate the antibody according to theinvention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating colorectalcancer, glioblastoma, gastric cancer, hepatocellular carcinoma, andrenal cell carcinoma, the efficacy of the therapeutic antibody can beassessed in various ways well known to the skilled practitioner. Forinstance, the size, number, and/or distribution of cancer in a subjectreceiving treatment may be monitored using standard tumor imagingtechniques. A therapeutically-administered antibody that arrests tumorgrowth, results in tumor shrinkage, and/or prevents the development ofnew tumors, compared to the disease course that would occurs in theabsence of antibody administration, is an efficacious antibody fortreatment of cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of a MHC class I epitope being used as an antigen, the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 268, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC: peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore, such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition, plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 268.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for, anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor, it is expressed. By “over-expressed” the inventorsmean that the polypeptide is present at a level at least 1.2-fold ofthat present in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore, anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from colorectalcancer, glioblastoma, gastric cancer, hepatocellular carcinoma, andrenal cell carcinoma, the medicament of the invention is preferably usedto treat colorectal cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, and renal cell carcinoma.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced, or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue ofcolorectal cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, and renal cell carcinoma patients with various HLA-A HLA-Band HLA-C alleles. It may contain MHC class I and MHC class II peptidesor elongated MHC class I peptides. In addition to the tumor associatedpeptides collected from several colorectal cancer, glioblastoma, gastriccancer, hepatocellular carcinoma, and renal cell carcinoma tissues, thewarehouse may contain HLA-A*02, HLA-A*01, HLA-A*03, HLA-A*24, HLA-B*07,HLA-B*08 and HLA-B*44 marker peptides. These peptides allow comparisonof the magnitude of T-cell immunity induced by TUMAPS in a quantitativemanner and hence allow important conclusion to be drawn on the capacityof the vaccine to elicit anti-tumor responses. Secondly, they functionas important positive control peptides derived from a “non-self” antigenin the case that any vaccine-induced T-cell responses to TUMAPs derivedfrom “self” antigens in a patient are not observed. And thirdly, it mayallow conclusions to be drawn, regarding the status of immunocompetenceof the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, colorectal cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, and renal cellcarcinoma samples from patients and blood from healthy donors wereanalyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue(colorectal cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, and renal cell carcinoma) compared with a range of normalorgans and tissues

3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.

4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs

5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.

6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromcolorectal cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, and renal cell carcinoma patients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multi-target approach. Intheory, an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients' tumor and,where possible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample, and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andover presentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from colorectal cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, and renal cell carcinoma cells and since itwas determined that these peptides are not or at lower levels present innormal tissues, these peptides can be used to diagnose the presence of acancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for colorectal cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, and renal cell carcinoma. Presence of groupsof peptides can enable classification or sub-classification of diseasedtissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

FIGURES

FIGS. 1A through 1P show exemplary expression profile of source genes ofthe present invention that are over-expressed in different cancersamples. Tumor (black dots) and normal (grey dots) samples are groupedaccording to organ of origin, and box-and-whisker plots representmedian, 25th and 75th percentile (box), and minimum and maximum(whiskers) RPKM values. Normal organs are ordered according to riskcategories. RPKM=reads per kilobase per million mapped reads. Normalsamples: blood cells; blood vessel; brain; heart; liver; lung; adipose:adipose tissue; adren. gl.: adrenal gland; bile duct; bladder; BM: bonemarrow; cartilage; esoph: esophagus; eye; gallb: gallbladder; head andneck; kidney; large_int: large intestine; LN: lymph node; nerve;pancreas; parathyr: parathyroid; perit: peritoneum; pituit: pituitary;skel. mus: skeletal muscle; skin; small_int: small intestine; spleen;stomach; thyroid; trachea; ureter; breast; ovary; placenta; prostate;testis; thymus; uterus. Tumor samples: CRC: colorectal cancer; GBM:glioblastoma; GC: gastric cancer; HCC: hepatocellular carcinoma; RCC:renal cell carcinoma. FIG. 1A) Gene symbol: EGFR, Peptide: LPSPTDSNFY(SEQ ID No.: 2), FIG. 1B) Gene symbol: PTPRZ1, Peptide: LTDYINANY (SEQID No.: 28), 1C) Gene symbol: CCDC146, Peptide: KMMALVAEL (SEQ ID No.:42), 1D) Gene symbol: MMP16, Peptide: YLPPTDPRMSV (SEQ ID No.: 54), 1E)Gene symbol: RP11-1220K2.2, Peptide: GLPDFVKEL (SEQ ID No.: 60), 1F)Gene symbol: GRM8, Peptide: ATMQSKLIQK (SEQ ID No.: 99), 1G) Genesymbol: HAVCR1, Peptide: GVIIAKKYFFK (SEQ ID No.: 101), 1H) Gene symbol:SEMA5B, Peptide: GTESGTILK (SEQ ID No.: 110), 1I) Gene symbol: PIWIL1,Peptide: SFDSNLLSF (SEQ ID No.: 133), 1J) Gene symbol: UGT1A3, Peptide:KYLSIPTVF (SEQ ID No.: 138), 1K) Gene symbol: FEZF1, Peptide: APAAVPSAPA(SEQ ID No.: 153), 1L) Gene symbol: MMP11, Peptide: RPASSLRP (SEQ IDNo.: 163), 1M) Gene symbol: QRFPR, Peptide: SPMWHVQQL (SEQ ID No.: 170),1N) Gene symbol: REG4, Peptide: SRSMRLLLL (SEQ ID No.: 190), 1O) Genesymbol: PTHLH, Peptide: AEIHTAEI (SEQ ID No.: 231), 1P) Gene symbol:EGFR, Peptide: DEYLIPQQGF (SEQ ID No.: 264).

FIG. 2 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*02+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAB and HLA-A*02 in complex withSeqID No 267 peptide (KTLGKLWRL, Seq ID NO: 267) (A, left panel). Afterthree cycles of stimulation, the detection of peptide-reactive cells wasperformed by 2D multimer staining with A*02/SeqID No 267 (A). Rightpanel (B) show control staining of cells stimulated with irrelevantA*02/peptide complexes. Viable singlet cells were gated for CD8+lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+ cells among CD8+ lymphocytes are indicated.

FIG. 3 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*24+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAB and HLA-A*24 in complex withSEQ ID NO: 268 peptide (A, left panel). After three cycles ofstimulation, the detection of peptide-reactive cells was performed by 2Dmultimer staining with A*24/SEQ ID NO: 268 (DYIPYVFKL, SEQ ID NO: 268)(A). Right panel (B) shows control staining of cells stimulated withirrelevant A*24/peptide complexes. Viable singlet cells were gated forCD8+ lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+ cells among CD8+ lymphocytes are indicated.

FIG. 4 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*01+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*01 in complex withSEQ ID NO: 6 peptide (VLDLSTNVY; A, left panel) and SEQ ID NO: 245peptide (ITEKNSGLY; B, left panel), respectively. After three cycles ofstimulation, the detection of peptide-reactive cells was performed by 2Dmultimer staining with A*01/SEQ ID NO: 6 (A) or A*01/SEQ ID NO: 245 (B).Right panels (A and B) show control staining of cells stimulated withirrelevant A*01/peptide complexes. Viable singlet cells were gated forCD8+ lymphocytes. Boolean gates helped excluding false-positive eventsdetected with multimers specific for different peptides. Frequencies ofspecific multimer+ cells among CD8+ lymphocytes are indicated.

FIG. 5 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*02+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*02 in complex withSEQ ID NO: 42 peptide (KMMALVAEL; A, left panel) and SEQ ID NO: 250peptide (KMILKMVQL; B, left panel), respectively. After three cycles ofstimulation, the detection of peptide-reactive cells was performed by 2Dmultimer staining with A*02/SEQ ID NO: 42 (A) or A*02/SEQ ID NO: 250(B). Right panels (A and B) show control staining of cells stimulatedwith irrelevant A*02/peptide complexes. Viable singlet cells were gatedfor CD8+ lymphocytes. Boolean gates helped excluding false-positiveevents detected with multimers specific for different peptides.Frequencies of specific multimer+ cells among CD8+ lymphocytes areindicated.

FIG. 6 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*03+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*03 in complex withSEQ ID NO: 94 peptide (KTYVGHPVKM; A, left panel) and SEQ ID NO: 110peptide (GTESGTILK; B, left panel), respectively. After three cycles ofstimulation, the detection of peptide-reactive cells was performed by 2Dmultimer staining with A*03/SEQ ID NO: 94 (A) or A*03/SEQ ID NO: 110(B). Right panels (A and B) show control staining of cells stimulatedwith irrelevant A*03/peptide complexes. Viable singlet cells were gatedfor CD8+ lymphocytes. Boolean gates helped excluding false-positiveevents detected with multimers specific for different peptides.Frequencies of specific multimer+ cells among CD8+ lymphocytes areindicated.

FIG. 7 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-A*24+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-A*24 in complex withSEQ ID NO: 138 peptide (KYLSIPTVF; left panel). After three cycles ofstimulation, the detection of peptide-reactive cells was performed by 2Dmultimer staining with A*02/SEQ ID NO: 138. Right panel shows controlstaining of cells stimulated with irrelevant A*24/peptide complexes.Viable singlet cells were gated for CD8+ lymphocytes. Boolean gateshelped excluding false-positive events detected with multimers specificfor different peptides. Frequencies of specific multimer+ cells amongCD8+ lymphocytes are indicated.

FIG. 8 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-B*07+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-B*07 in complex withSEQ ID NO: 170 peptide (SPMWHVQQL; A, left panel) and SEQ ID NO: 155peptide (FPYPYAERL; B, left panel), respectively. After three cycles ofstimulation, the detection of peptide-reactive cells was performed by 2Dmultimer staining with B*07/SEQ ID NO: 170 (A) or B*07/SEQ ID NO: 155(B). Right panels (A and B) show control staining of cells stimulatedwith irrelevant B*07/peptide complexes. Viable singlet cells were gatedfor CD8+ lymphocytes. Boolean gates helped excluding false-positiveevents detected with multimers specific for different peptides.Frequencies of specific multimer+ cells among CD8+ lymphocytes areindicated.

FIG. 9 shows exemplary results of peptide-specific in vitro CD8+ T cellresponses of a healthy HLA-B*44+ donor. CD8+ T cells were primed usingartificial APCs coated with anti-CD28 mAb and HLA-B*44 in complex withSEQ ID NO: 225 peptide (SEAFPSRAL; A, left panel) and SEQ ID NO: 236peptide (EEKLIIQDF; B, left panel), respectively. After three cycles ofstimulation, the detection of peptide-reactive cells was performed by 2Dmultimer staining with B*44/SEQ ID NO: 225 (A) or B*44/SEQ ID NO: 236(B). Right panels (A and B) show control staining of cells stimulatedwith irrelevant B*44/peptide complexes. Viable singlet cells were gatedfor CD8+ lymphocytes. Boolean gates helped excluding false-positiveevents detected with multimers specific for different peptides.Frequencies of specific multimer+ cells among CD8+ lymphocytes areindicated.

EXAMPLES Example 1

Identification of Tumor Associated Peptides Presented on the CellSurface

Tissue Samples

Patients' tumor tissues and normal tissues were obtained from theUniversity Hospital Tübingen (Tübingen, Germany). Written informedconsents of all patients had been given before surgery or autopsy.Tissues were shock-frozen immediately after excision and stored untilisolation of TUMAPs at −70° C. or below. Sample numbers for TUMAPselection were: for renal cancer N=79, for colorectal cancer N=35, forhepatocellular carcinoma N=22, for gastric cancer N=10, and forglioblastoma N=12.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, -B, C-specific antibodyW6/32, the HLA-DR specific antibody L243 and the pan-HLA class IIspecific antibody Tü39, CNBr-activated sepharose, acid treatment, andultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (Ultimate 3000 RSLC NanoUHPLC System, Dionex) and the eluting peptides were analyzed inLTQ-Orbitrap and Fusion Lumos hybrid mass spectrometers (ThermoElectron)equipped with an ESI source. Peptide samples were loaded with 3% ofsolvent B (20% H₂O, 80% acetonitrile and 0.04% formic acid) on a 2 cmPepMap 100 C18 Nanotrap column (Dionex) at a flowrate of 4 μl/min for 10min. Separation was performed on either 25 cm or 50 cm PepMap C18columns with a particle size of 2 μm (Dionex) mounted in a column ovenrunning at 50° C. The applied gradient ranged from 3 to 32% solvent Bwithin 90 min at a flow rate of 300 nl/min (for 25 cm columns) or 140min at a flow rate of 175 nl/min (for 50 cm columns). (Solvent A: 99%H₂O, 1% ACN and 0.1% formic acid; Solvent B: 20% H₂O, 80% ACN and 0.1%formic acid).

Mass spectrometry analysis was performed in data dependent acquisitionmode employing a top five method (i.e. during each survey scan the fivemost abundant precursor ions were selected for fragmentation).Alternatively, a TopSpeed method was employed for analysis on FusionLumos instruments.

Survey scans were recorded in the Orbitrap at a resolution of 60,000(for Orbitrap XL) or 120,000 (for Orbitrap Fusion Lumos). MS/MS analysiswas performed by collision induced dissociation (CID, normalizedcollision energy 35%, activation time 30 ms, isolation width 1.3 m/z)with subsequent analysis in the linear trap quadrupole (LTQ). Mass rangefor HLA class I ligands was limited to 400-650 m/z with possible chargestates 2+ and 3+ selected for fragmentation. For HLA class II mass rangewas set to 300-1500 m/z allowing for fragmentation with all positivecharge states ≥2.

Tandem mass spectra were interpreted by MASCOT or SEQUEST at a fixedfalse discovery rate (q≤0.05) and additional manual control. In caseswhere the identified peptide sequence was uncertain it was additionallyvalidated by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide.

Table 8a and 8b show the presentation on various cancer entities forselected peptides, and thus the particular relevance of the peptides asmentioned for the diagnosis and/or treatment of the cancers as indicated(e.g. peptide SEQ ID No. 1 for colorectal cancer (Table 8a) and for GBCand GC (Table 8b), peptide SEQ ID No. 50 for colorectal cancer andhepatocellular carcinoma (Table 8a) and for CCC, GBM, HNSCC, NHL,NSCLCsquam, PACA, SCLC and UBC (Table 8b)).

TABLE 8a  Overview of presentation of selected tumor-associated peptides of the present inventionacross exemplary and preferred entities (diseases).GBM = glioblastoma, CRC = colorectal cancer, RCC =renal cell carcinoma, HCC = hepatocellularcarcinoma, GC = gastric cancer. SEQ ID Peptide Presentation  No.Sequence on cancer entities 1 RSDPVTLDV CRC 2 LPSPTDSNFY RCC 3ASSTDSASYY HCC 4 NSDLKYNAL HCC 5 SILGSDVRVPSY HCC 6 VLDLSTNVY HCC 7LITGDPKAAYDY CRC 8 TPVTEFSLNTY CRC 9 FITAQNHGY HCC 10 ITAQNHGY HCC 11LSAGSGPGQY HCC 12 ITFGERFEY RCC 13 GSTMVEHNY RCC 14 YTERDGSAMVY GBM 15LTDYLKNTY HCC 16 LSLIDRLVLY RCC 17 YTDKLQHY CRC 18 EVSNGKWLLY RCC 19VSNGKWLLY RCC 20 STDEITTRY HCC 21 STDIGALMY CRC 22 TLEQVQLYY CRC 23TASEDVFQY CRC 24 YTHHLFIFY CRC 25 LMKEVMEHY RCC 26 EVLDSHIHAY GBM 27LDSHIHAY GBM 28 LTDYINANY GBM 29 SVTDLEMPHY GBM 30 VLDSHIHAY GBM 31VTDLEMPHY GBM 32 ATVGYFIFY RCC 33 FADKIHLAY RCC 34 ITDFNNIRY CRC 35FASDLLHLY HCC 36 YAAYIIHAY RCC 37 LTDSFPLKV HCC 38 VMLNSNVLL GBM 39YLLPSVVLL GBM 40 KIDDIWNLEV HCC 41 SLQDTKITL HCC 42 KMMALVAEL RCC 43GLMTIVTSL CRC 44 SQTGFVVLV GBM 45 KLLDEVTYL RCC 46 VLITGLPLI RCC 47YQDSWFQQL RCC 48 NLTFIIILI HCC 49 NLASRPYSL HCC 50 ELMPRVYTL CRC, HCC 51ALAAELNQL GBM 52 YVSSGEMMV GBM 53 LLMTSLTES GBM 54 YLPPTDPRMSV GBM 55RLWQIQHHL RCC 56 FLNQIYTQL GC 57 GLTGVIMTI CRC 58 MLCLLLTL RCC 59KLHEIYIQA GBM 60 GLPDFVKEL GC 61 RLFGLFLNNV RCC 62 GSYSALLAKK HCC 63KVLGPNGLLK HCC 64 STTKLYLAK HCC 65 VLGPNGLLK HCC 66 ATYEGIQKK HCC 67ATALSLSNK HCC 68 ATAYGSTVSK HCC 69 ATAYGSTVSKR HCC 70 ATWSASLKNK HCC 71KLGNNPVSK HCC 72 KQVFPGLNY HCC 73 KSFDRHFEK HCC 74 QLYSKFLLK HCC 75QVPTFTIPK HCC 76 SAFGYVFPK HCC 77 SSASLAHMK HCC 78 STKSTSPPK HCC 79STNNEGNLK HCC 80 STSHHLVSR HCC 81 SVKLQGTSK HCC 82 TAYGSTVSK HCC 83TAYGSTVSKR HCC 84 TVASLHTEK HCC 85 KMAAWPFSR HCC 86 KTPSGALHRK HCC 87SSYSRSSAVK GBM 88 MLLQQPLIY RCC 89 KITDFGLAK RCC 90 GSRLGKYYVK RCC 91SLIDRLVLY RCC 92 AVLDLGSLLAK RCC 93 ALDKPGKSK GBM 94 KTYVGHPVKM RCC 95RLFESSFHY RCC 96 FSLAGALNAGFK GBM 97 RMPPPLPTR GBM 98 KLYPTYSTK HCC 99ATMQSKLIQK RCC 100 ALLGVIIAK RCC 101 GVIIAKKYFFK RCC 102 IIAKKYFFK RCC103 KSWTASSSY RCC 104 STQDTLLIK CRC 105 GSAALYLLR RCC 106 RLSPNDQYK RCC107 EIYGGHHAGF GBM 108 LLKSSVGNFY GBM 109 KIIAPLVTR RCC 110 GTESGTILKRCC 111 KIKEHVRSK HCC 112 KMMADYGIRK HCC 113 VWAKILSAF HCC 114 KFLDSNIKFHCC 115 YFEEAANFL HCC 116 LVLDYSKDYNHW HCC 117 NFLPPIIARF RCC 118TYISKTIAL RCC 119 YMKALGVGF RCC 120 MYAKEFDLL HCC 121 SYIEKVRFL GBM 122KLYGMPTDFGF GC 123 RQYLAINQI HCC 124 EVYSPEADQW GBM 125 IYGPKYIHPSF GC126 TFQDKTLNF CRC 127 IFINLSPEF GC 128 SYTKVEARL GC 129 VFLNQIYTQL GC130 VYGDGHYLTF GC 131 KQLDHNLTF CRC 132 VYNPVIYVF HCC 133 SFDSNLLSF CRC134 TYLTGRQF CRC 135 VIAPIISNF CRC 136 EYNNIQHLF RCC 137 KYLSLSNSF RCC138 KYLSIPTVF HCC 139 PYASLASELF HCC 140 KYLSIPAVF HCC 141 KYLSIPAVFFHCC 142 SSFPGAGNTW GBM 143 FELPTGAGLQL HCC 144 IPEPSAQQL HCC 145RVPSYTLIL HCC 146 SPGDKRLAA HCC 147 SPIKVPLLL HCC 148 VPDGVSKVL HCC 149YPLTGDTRL HCC 150 KPSSKALGTSL CRC 151 VVHPRTLLL RCC 152 IPSRLLAIL RCC153 APAAVPSAPA GBM 154 GPGTRLSL GBM 155 FPYPYAERL CRC 156 HPQVVILSL RCC157 SPSPGKDPTL RCC 158 VPERGEPEL RCC 159 FPAHPSLLL RCC 160 RPAPADSAL RCC161 NPYEGRVEV RCC 162 MPMISIPRV RCC 163 RPASSLRP CRC, HCC 164ISTPSEVSTPL GC 165 TPIAKVSEL CRC 166 HDPDVGSNSL GBM 167 YPSEVEHMF RCC168 IPTDKLLVI RCC 169 FPTEVTPHAF GBM 170 SPMWHVQQL RCC 171 APKLFAVAF RCC172 KPAHYPLIAL RCC 173 MVPSAGQLALF RCC 174 VPSLQRLML RCC 175 HPIETLVDIFRCC 176 AAMSRYEL HCC 177 DLKYNALDL HCC 178 HAKEKLTAL HCC 179IQIYKKLRTSSF HCC 180 LLKAEPLAF HCC 181 YKKLRTSSF HCC 182 LPFLRENDL GBM183 FQKLKLLSL CRC 184 EPVKKSRL RCC 185 NPNLKTLL GBM 186 SLIDRLVL RCC 187YVKERSKAM RCC 188 SALDHVTRL RCC 189 HIFLRTTL HCC 190 SRSMRLLLL GC 191LINLKYLSL RCC 192 LPMLKVLNL RCC 193 LSYNKYLQL RCC 194 EAKRHLLQV HCC 195AEAVLKTLQEL HCC 196 AEQTGTWKL HCC 197 EEAKQVLFL HCC 198 FELPTGAGL HCC199 GEATLQRIY HCC 200 GEELGFASL HCC 201 GEHTSKATL HCC 202 KEFNLQNMGL HCC203 KENFAGEATL HCC 204 KESQLPTVM HCC 205 QEVLLQTFL HCC 206 SEPINIIDALHCC 207 TEATMTFKY HCC 208 AEHDAVRNAL CRC 209 YEVDTVLRY GBM 210 SENIVIQVYHCC 211 TEKEMIQKL RCC 212 AEETCAPSV RCC 213 TTMDQKSLW GBM 214 AEQPDGLILHCC 215 AFITAQNHGY HCC 216 LQEEKVPAIY HCC 217 NEINEKIAPSF HCC 218AEGGKVPIKW GBM, RCC 219 AENAEYLRV GBM 220 KEITGFLLI GBM 221 AEERAEAKKKFRCC 222 NEISTFHNL HCC 223 SEVPVARVW HCC 224 SESAVFHGF RCC 225 SEAFPSRALRCC 226 EELLHGQLF GC 227 TEHTQSQAAW RCC 228 AEKQTPDGRKY GBM 229KESDGFHRF RCC 230 AENLFRAFL CRC 231 AEIHTAEI RCC 232 AEKDGKLTDY GBM 233DESEKTTKSF GBM 234 EEESLLTSF GBM 235 EEFETLKEF GBM 236 EEKLIIQDF GBM 237LEMPHYSTF GBM 238 SENPETITY GBM 239 TEVLDSHIHAY GBM 240 HELENHSMY GBM241 REAEPIPKM GBM 242 FSDKELAAY HCC 243 RSPNNFLSY RCC 244 RSDPVTLNV CRC245 ITEKNSGLY CRC 246 YSDLHAFYY GBM 247 RSDPGGGGLAY GBM 248 YSHAAGQGTGLYCRC 249 ALFPERITV GBM 250 KMILKMVQL RCC 251 RLASRPLLL GBM 252 RIYNGIGVSRRCC 253 KLFGTSGQK GBM 254 AVATKFVNK GBM 255 LPDGSRVEL CRC 256 LPALPQQLICRC 257 SPLRGGSSL CRC 258 APSGTRVVQVL GBM 259 RPAVGHSGL HCC 260EEAPLVTKAF HCC 261 IEALLESSL RCC 262 MELLLVNKL RCC 263 QQATPGPAY CRC 264DEYLIPQQGF RCC 265 EEVDVPIKLY CRC 266 ARLTPIPFGL HCC

TABLE 8bOverview of presentation of selected tumor-associated peptides of the presentinvention across entities (diseases).BRCA = breast cancer, CCC = bile duct cancer, GBM = brain cancer, CRC = colorectalcarcinoma, OSCAR = esophageal cancer, GBC = gallbladder adenocarcinoma, GC =gastric cancer, HNSCC = head and neck squamous cell carcinoma, HCC = hepatocellularcarcinoma, MEL = melanoma, NHL = non-Hodgkin lymphoma, NSCLCadeno = non-smallcell lung cancer adenocarcinoma, NSCLCother = NSCLC samples that could notunambiguously be assigned to NSCLCadeno or NSCLCsquam, NSCLCsquam =squamous cell non-small cell lung cancer, OC = ovarian cancer, PACA = pancreaticcancer, PRCA = prostate cancer and benign prostate hyperplasia, RCC = renal cellcarcinoma, SCLC = small cell lung cancer, UBC = urinary bladder cancer, UEC = uterinecancer. SEQ ID No. Sequence Peptide Presentation on tumor types 1RSDPVTLDV GBC, GC 2 LPSPTDSNFY CCC, OSCAR 5 SILGSDVRVPSYNSCLCadeno, PACA 6 VLDLSTNVY CCC, GBC 8 TPVTEFSLNTYBRCA, CCC, GBC, GC, HNSCC, MEL, NSCLCadeno,NSCLCsquam, OC, OSCAR, PACA, RCC, SCLC, UBC, UEC 9 FITAQNHGY GC 10ITAQNHGY GC 12 ITFGERFEY CRC, GC, HCC, HNSCC, MEL, OSCAR, PACA, PRCA,UBC 13 GSTMVEHNY GBC, PRCA 14 YTERDGSAMVY BRCA, SCLC, UEC 15 LTDYLKNTYGBC, GC, NSCLCadeno, NSCLCsquam, PRCA, RCC, UEC 16 LSLIDRLVLYHCC, NSCLCsquam, UEC 17 YTDKLQHY BRCA, HNSCC, NSCLCadeno, SCLC 21STDIGALMY BRCA, GBC, GC, GEJC, HNSCC, MEL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, RCC, SCLC, UBC 22 TLEQVQLYY GC25 LMKEVMEHY MEL, NSCLCadeno, NSCLCsquam, OC, SCLC 26 EVLDSHIHAYBRCA, CRC, GC, HNSCC, MEL, NHL, NSCLCsquam, OSCAR, PACA, PRCA, SCLC, UBC27 LDSHIHAY MEL 28 LTDYINANY GC, MEL, NSCLCsquam, OSCAR 29 SVTDLEMPHYNSCLCsquam, OSCAR 30 VLDSHIHAY GC, HNSCC, MEL, NSCLCsquam, OSCAR 31VTDLEMPHY GC, HNSCC, MEL, NSCLCsquam, OSCAR 33 FADKIHLAY GBC, GC, OSCAR34 ITDFNNIRY BRCA, GC, GEJC, HNSCC, MEL, NHL, OSCAR 36 YAAYIIHAYHNSCC, NSCLCadeno, NSCLCsquam, OC, OSCAR, UEC 37 LTDSFPLKV CCC 39YLLPSVVLL AML, BRCA, CCC, CLL, CRC, GBC, GC, GEJC, HCC,HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 42 KMMALVAEL OC44 SQTGFVVLV AML, BRCA, CCC, CLL, CRC, GBC, GC, GEJC, HCC,HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 45 KLLDEVTYLBRCA, CCC, CRC, GBC, GBM, GC, GEJC, HCC, HNSCC,MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam,OC, OSCAR, PACA, PRCA, SCLC, UBC, UEC 46 VLITGLPLIBRCA, CCC, CRC, HCC, HNSCC, NSCLCsquam, UBC 47 YQDSWFQQL HCC, UBC 50ELMPRVYTL CCC, GBM, HNSCC, NHL, NSCLCsquam, PACA, SCLC, UBC 51 ALAAELNQLNHL 53 LLMTSLTES AML, BRCA, CLL, CRC, GBC, GC, HCC, HNSCC, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, PACA, PRCA, SCLC, UBC, UEC 56FLNQIYTQL CRC, GEJC, PACA 60 GLPDFVKEL GBC, MEL, OSCAR 67 ATALSLSNK CCC68 ATAYGSTVSK CCC, CRC, GBC, PACA 70 ATWSASLKNK CCC 72 KQVFPGLNYCCC, CRC, RCC, SCLC 74 QLYSKFLLK CCC, OSCAR, RCC, SCLC 75 QVPTFTIPKAML, CCC, OSCAR, SCLC 76 SAFGYVFPKAML, BRCA, CCC, CRC, GBC, GC, MEL, NSCLCother,OSCAR, PACA, PRCA, RCC, SCLC, UBC 82 TAYGSTVSK CCC, NHL 85 KMAAWPFSRBRCA, GBC, NSCLCadeno, NSCLCsquam, OC 89 KITDFGLAK GBM 90 GSRLGKYYVKGBM, HCC, HNSCC, NSCLCadeno, NSCLCother,NSCLCsquam, OC, PACA, SCLC, UBC, UEC 91 SLIDRLVLYGBC, NSCLCadeno, NSCLCsquam, PACA, SCLC 92 AVLDLGSLLAKBRCA, CCC, CRC, GBC, GBM, GC, HCC, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR,PACA, PRCA, SCLC, UBC, UEC 94 KTYVGHPVKMCRC, GBC, HNSCC, MEL, NSCLCadeno, PACA 98 KLYPTYSTK OSCAR, SCLC 99ATMQSKLIQK SCLC 100 ALLGVIIAK CCC, CRC, NSCLCadeno, OC, UBC, UEC 103KSWTASSSY CRC 104 STQDTLLIK BRCA, GBC, GC, HNSCC, NHL, NSCLCsquam, OC,OSCAR, PACA, RCC, SCLC, UBC, UEC 105 GSAALYLLR NSCLCsquam, UBC, UEC 107EIYGGHHAGF SCLC 109 KIIAPLVTR HCC, NSCLCadeno, OC, UEC 112 KMMADYGIRKBRCA, CCC, CRC, GBC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, RCC, UEC 113 VWAKILSAFCLL, GBC, NHL 115 YFEEAANFL CCC, GBC 117 NFLPPIIARFCRC, GBC, GBM, HNSCC, MEL, NSCLCadeno, OC, OSCAR, UEC 118 TYISKTIALBRCA, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR,PACA, PRCA, SCLC, UBC, UEC 119 YMKALGVGF GBM 120 MYAKEFDLLBRCA, CLL, CRC, GBC, GC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA, PRCA, RCC, UEC 121SYIEKVRFL BRCA, NHL, SCLC 122 KLYGMPTDFGFBRCA, CRC, HNSCC, NHL, NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC, UBC 123RQYLAINQI BRCA, CCC, CLL, CRC, GBC, GBM, GC, HNSCC, MEL,NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, RCC, SCLC, UBC 124 EVYSPEADQW OSCAR 125 IYGPKYIHPSFGBC, NSCLCadeno 126 TFQDKTLNF GC, MEL, OSCAR, PRCA, SCLC, UBC, UEC 127IFINLSPEF CCC, GBC, NSCLCadeno, PACA 128 SYTKVEARLCCC, CRC, GBC, NSCLCadeno, PACA 129 VFLNQIYTQL GBC, PACA 130 VYGDGHYLTFNSCLCadeno, PACA 132 VYNPVIYVFAML, BRCA, CCC, CLL, CRC, GBC, GBM, GC, HNSCC,MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam,OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 134 TYLTGRQFGC, OSCAR, PRCA, SCLC, UEC 135 VIAPIISNFAML, BRCA, CCC, CLL, GBC, GBM, GC, HCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, PACA, PRCA, RCC, SCLC, UEC 136EYNNIQHLF GBC, GBM, GC, HCC, NSCLCadeno, NSCLCother,NSCLCsquam, OC, PRCA, UBC, UEC 137 KYLSLSNSF HCC, OC, PACA, PRCA 138KYLSIPTVF NSCLCadeno 139 PYASLASELF GC 140 KYLSIPAVF CCC, GBC, GC, HNSCC141 KYLSIPAVFF GBC, GC, HNSCC, SCLC 142 SSFPGAGNTW OSCAR, SCLC 144IPEPSAQQL CCC, CRC, GBC, NHL 145 RVPSYTLIL NSCLCsquam, PRCA 147SPIKVPLLL GBC, GC, OSCAR, PACA, RCC 148 VPDGVSKVL CCC 150 KPSSKALGTSLGBC 151 VVHPRTLLL CCC, HCC, UEC 155 FPYPYAERLGC, HNSCC, NSCLCsquam, OC, OSCAR, PACA 156 HPQVVILSL GBC 157 SPSPGKDPTLNSCLCadeno, PRCA 159 FPAHPSLLL NSCLCadeno 160 RPAPADSAL GBC, NSCLCadeno161 NPYEGRVEV CRC, GBC, GBM, GC, MEL 162 MPMISIPRV GBM, NHL 163 RPASSLRPOC 165 TPIAKVSEL HCC, OC, UEC 166 HDPDVGSNSLBRCA, CRC, GBC, GC, HCC, HNSCC, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR,PACA, PRCA, SCLC, UBC, UEC 167 YPSEVEHMFMEL, NSCLCadeno, NSCLCother, NSCLCsquam, PRCA, UEC 168 IPTDKLLVIBRCA, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, SCLC, UBC, UEC 169FPTEVTPHAF HNSCC, MEL, NSCLCsquam, SCLC 171 APKLFAVAF NSCLCadeno 173MVPSAGQLALF GC, HNSCC, OSCAR, SCLC 174 VPSLQRLML CRC, NSCLCadeno 175HPIETLVDIF CCC, CRC, GBC, GBM, GC, MEL, NSCLCadeno,NSCLCsquam, OC, SCLC, UBC, UEC 177 DLKYNALDL CCC 178 HAKEKLTAL CCC 180LLKAEPLAF CCC, GBC, RCC, SCLC 182 LPFLRENDL PRCA, UEC 183 FQKLKLLSLGBC, PACA 188 SALDHVTRL NHL, PRCA 190 SRSMRLLLL CRC 192 LPMLKVLNLHNSCC, NSCLCadeno, NSCLCother, NSCLCsquam, OC 193 LSYNKYLQL HCC, UEC 194EAKRHLLQV CCC, MEL, NHL, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, SCLC195 AEAVLKTLQEL CCC 196 AEQTGTWKL CCC 197 EEAKQVLFL RCC 198 FELPTGAGLCCC, HNSCC, RCC, SCLC 199 GEATLQRIY CCC, CRC 200 GEELGFASLCCC, RCC, SCLC 206 SEPINIIDAL CCC, RCC, SCLC 207 TEATMTFKY CCC, GBC 208AEHDAVRNAL GC, HNSCC 209 YEVDTVLRY BRCA, MEL 210 SENIVIQVYCCC, MEL, NSCLCother 212 AEETCAPSV BRCA, MEL, UEC 214 AEQPDGLILCCC, SCLC 218 AEGGKVPIKW HNSCC, NSCLCadeno, PRCA, UEC 219 AENAEYLRV RCC220 KEITGFLLI NSCLCadeno, UEC 223 SEVPVARVWCCC, CRC, GBC, NSCLCadeno, OSCAR, RCC 224 SESAVFHGFBRCA, CCC, CRC, GBC, GBM, GC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC, UBC, UEC 226EELLHGQLF CCC, CRC, NSCLCadeno, OSCAR, PACA 227 TEHTQSQAAWCLL, HCC, HNSCC, NSCLCadeno, UEC 229 KESDGFHRFBRCA, CCC, CRC, GBC, GBM, GC, HCC, HNSCC, MEL,NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, SCLC, UBC, UEC230 AENLFRAFL AML, CCC, CLL, GBC, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, UBC, UEC 232AEKDGKLTDY MEL 234 EEESLLTSF NSCLCsquam, OSCAR 235 EEFETLKEF HNSCC, MEL236 EEKLIIQDF HNSCC, MEL, NSCLCadeno, NSCLCsquam, OSCAR, PRCA 238SENPETITY HNSCC 239 TEVLDSHIHAY MEL, NSCLCsquam, OSCAR 240 HELENHSMYOSCAR 242 FSDKELAAY BRCA, CCC, GBC, GBM, GC, MEL, NHL, NSCLCadeno,NSCLCsquam, OSCAR, PACA 243 RSPNNFLSY GBC 244 RSDPVTLNVBRCA, GBC, GC, NSCLCadeno, PACA 245 ITEKNSGLY GBC, GC 246 YSDLHAFYYAML, BRCA, CLL, CRC, GBC, GC, GEJC, HCC, HNSCC,MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam,OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 247 RSDPGGGGLAYAML, BRCA, CRC, GBC, GC, GEJC, HCC, HNSCC, MEL,NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, SCLC, UBC, UEC 248 YSHAAGQGTGLYBRCA, GBC, HNSCC, OSCAR, UEC 249 ALFPERITVAML, BRCA, CCC, GBC, GBM, GC, HCC, HNSCC, MEL,NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 251 RLASRPLLLNSCLCsquam, OC, OSCAR 252 RIYNGIGVSR CRC, GBM, MEL, NSCLCadeno 254AVATKFVNK AML, BRCA, CCC, CRC, GBC, GC, HCC, HNSCC, MEL,NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 255 LPDGSRVEL BRCA 256 LPALPQQLIGBC, GBM, GC, MEL, NHL, NSCLCadeno, NSCLCsquam, OSCAR, PACA, SCLC, UEC257 SPLRGGSSL AML, BRCA, GC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCsquam, OC, OSCAR, SCLC, UBC 258 APSGTRVVQVLBRCA, CCC, CRC, GBC, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR,PACA, PRCA, RCC, SCLC, UBC, UEC 259 RPAVGHSGLAML, BRCA, HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OC, OSCAR, RCC 260EEAPLVTKAF AML, CLL, CRC, MEL, NHL, NSCLCadeno, NSCLCsquam, PRCA, UEC261 IEALLESSL BRCA, CCC, CLL, CRC, GBC, GBM, GC, HCC, HNSCC,MEL, NHL, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC, UBC, UEC262 MELLLVNKL HCC, HNSCC, MEL, OC, UEC 263 QQATPGPAYAML, BRCA, CCC, GBC, GC, NSCLCadeno, NSCLCother,NSCLCsquam, OSCAR, PACA, UBC 264 DEYLIPQQGFBRCA, CCC, CRC, GBC, GBM, GC, HCC, HNSCC, MEL,NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, SCLC, UBC, UEC 265 EEVDVPIKLYBRCA, CCC, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR,PACA, PRCA, RCC, SCLC, UEC 266 ARLTPIPFGLBRCA, GBM, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, RCC, SCLC, UBC, UEC

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNEASY® (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK); Bio-OptionsInc. (Brea, Calif., USA); Geneticist Inc. (Glendale, Calif., USA);ProteoGenex Inc. (Culver City, Calif., USA); Tissue Solutions Ltd(Glasgow, UK). Total RNA from tumor tissues for RNASeq experiments wasobtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK);BioCat GmbH (Heidelberg, Germany); BioServe (Beltsville, Md., USA);Geneticist Inc. (Glendale, Calif., USA); Istituto Nazionale Tumori“Pascale” (Naples, Italy); ProteoGenex Inc. (Culver City, Calif., USA);University Hospital Heidelberg (Heidelberg, Germany). Quality andquantity of all RNA samples were assessed on an Agilent 2100 Bioanalyzer(Agilent, Waldbronn, Germany) using the RNA 6000 Pico LabChip Kit(Agilent).

RNAseq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (IlluminaInc., San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolar and sequenced on the Illumina HiSeq2500 sequencer according to the manufacturer's instructions, generating50 bp single end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of the ensemblsequence database (Ensembl77). Exon reads are normalized for exon lengthand alignment size to obtain RPKM values.

Exemplary expression profiles of source genes of the present inventionthat are highly over-expressed or exclusively expressed in colorectalcancer, glioblastoma, gastric cancer, hepatocellular carcinoma, andrenal cell carcinoma are shown in FIG. 1. Expression scores for furtherexemplary genes are shown in Table 9.

TABLE 9 Expression scores. The table lists peptides from genes that are very highly over-expressed in tumors compared to a panel of normal tissues (+++), highly over-expressedin tumors compared to a panel of normal tissues (++) or over-expressed in tumorscompared to a panel of normal tissues (+). The baseline for this score was calculated frommeasurements of the following relevant normal tissues: blood cells, blood vessels, brain,heart, liver, lung, adipose tissue, adrenal gland, bile duct, bladder, bone marrow,cartilage, esophagus, eye, gallbladder, head and neck, kidney, large intestine, lymphnode, nerve, pancreas, parathyroid, peritoneum, pituitary, pleura, skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea, ureter. In case expression data for several samples of the same tissue type were available, the arithmetic mean of all respective samples  was used for the calculation.SEQ ID Gene Expression No Sequence CRC GBM GC HCC RCC 1 RSDPVTLDV + 2LPSPTDSNFY +++ + 3 ASSTDSASYY ++ 4 NSDLKYNAL + 5 SILGSDVRVPSY + 6VLDLSTNVY ++ 7 LITGDPKAAYDY + + + 8 TPVTEFSLNTY + + 9 FITAQNHGY + 10ITAQNHGY + 11 LSAGSGPGQY + 12 ITFGERFEY + 13 GSTMVEHNY + + 14YTERDGSAMVY + 15 LTDYLKNTY + 16 LSLIDRLVLY + 17 YTDKLQHY + 18EVSNGKWLLY + 19 VSNGKWLLY + 20 STDEITTRY ++ 21 STDIGALMY + ++ 22TLEQVQLYY + 23 TASEDVFQY + 24 YTHHLFIFY ++ 25 LMKEVMEHY + 26 EVLDSHIHAY++ 27 LDSHIHAY ++ 28 LTDYINANY +++ 29 SVTDLEMPHY +++ 30 VLDSHIHAY ++ 31VTDLEMPHY +++ 32 ATVGYFIFY + + 33 FADKIHLAY ++ + 34 ITDFNNIRY 35FASDLLHLY +++ 36 YAAYIIHAY + 37 LTDSFPLKV + 38 VMLNSNVLL + 39YLLPSVVLL + 40 KIDDIWNLEV + 41 SLQDTKITL + 42 KMMALVAEL ++ 43 GLMTIVTSL++ 44 SQTGFVVLV + 45 KLLDEVTYL + 46 VLITGLPLI + 47 YQDSWFQQL + 48NLTFIIILI +++ 49 NLASRPYSL + 50 ELMPRVYTL + + + + + 51 ALAAELNQL + 52YVSSGEMMV + 53 LLMTSLTES + 54 YLPPTDPRMSV +++ 55 RLWQIQHHL + 56FLNQIYTQL + 57 GLTGVIMTI ++ 58 MLCLLLTL ++ + 59 KLHEIYIQA + 60 GLPDFVKEL+++ ++ 61 RLFGLFLNNV + 62 GSYSALLAKK + 63 KVLGPNGLLK + 64 STTKLYLAK + 65VLGPNGLLK + 66 ATYEGIQKK + 67 ATALSLSNK + 68 ATAYGSTVSK ++ 69ATAYGSTVSKR ++ 70 ATWSASLKNK + 71 KLGNNPVSK + 72 KQVFPGLNY ++ 73KSFDRHFEK + 74 QLYSKFLLK + 75 QVPTFTIPK ++ 76 SAFGYVFPK + 77 SSASLAHMK +78 STKSTSPPK ++ 79 STNNEGNLK + 80 STSHHLVSR + 81 SVKLQGTSK + 82TAYGSTVSK ++ 83 TAYGSTVSKR ++ 84 TVASLHTEK + 85 KMAAWPFSR + 86KTPSGALHRK + 87 SSYSRSSAVK + 88 MLLQQPLIY + 89 KITDFGLAK +++ + 90GSRLGKYYVK + 91 SLIDRLVLY + 92 AVLDLGSLLAK + 93 ALDKPGKSK + 94KTYVGHPVKM + + + + + 95 RLFESSFHY ++ 96 FSLAGALNAGFK + 97 RMPPPLPTR + 98KLYPTYSTK + 99 ATMQSKLIQK +++ + 100 ALLGVIIAK ++ 101 GVIIAKKYFFK ++ 102IIAKKYFFK ++ 103 KSWTASSSY + + + 104 STQDTLLIK + + 105 GSAALYLLR ++ 106RLSPNDQYK ++ 107 EIYGGHHAGF ++ 108 LLKSSVGNFY + 109 KIIAPLVTR + 110GTESGTILK ++ +++ 111 KIKEHVRSK + 112 KMMADYGIRK + 113 VWAKILSAF + 114KFLDSNIKF + 115 YFEEAANFL + 116 LVLDYSKDYNHW + 117 NFLPPIIARF + 118TYISKTIAL + 119 YMKALGVGF + + 120 MYAKEFDLL + 121 SYIEKVRFL + 122KLYGMPTDFGF + 123 RQYLAINQI + 124 EVYSPEADQW +++ 125 IYGPKYIHPSF ++ +126 TFQDKTLNF ++ + 127 IFINLSPEF + 128 SYTKVEARL + 129 VFLNQIYTQL + 130VYGDGHYLTF + 131 KQLDHNLTF ++ 132 VYNPVIYVF + 133 SFDSNLLSF +++ + 134TYLTGRQF + 135 VIAPIISNF + 136 EYNNIQHLF + 137 KYLSLSNSF + 138 KYLSIPTVF+++ 139 PYASLASELF + 140 KYLSIPAVF + 141 KYLSIPAVFF + 142 SSFPGAGNTW +143 FELPTGAGLQL ++ 144 IPEPSAQQL ++ 145 RVPSYTLIL + 146 SPGDKRLAA ++ 147SPIKVPLLL + 148 VPDGVSKVL ++ 149 YPLTGDTRL ++ 150 KPSSKALGTSL + 151VVHPRTLLL + 152 IPSRLLAIL + 153 APAAVPSAPA ++ ++ +++ + 154 GPGTRLSL +155 FPYPYAERL ++ ++ 156 HPQVVILSL ++ 157 SPSPGKDPTL + 158 VPERGEPEL +159 FPAHPSLLL + 160 RPAPADSAL +++ 161 NPYEGRVEV + 162 MPMISIPRV + 163RPASSLRP ++ +++ + 164 ISTPSEVSTPL + 165 TPIAKVSEL + +++ 166 HDPDVGSNSL +167 YPSEVEHMF + 168 IPTDKLLVI + 169 FPTEVTPHAF +++ 170 SPMWHVQQL +++ 171APKLFAVAF + 172 KPAHYPLIAL + ++ 173 MVPSAGQLALF ++ 174 VPSLQRLML + 175HPIETLVDIF + 176 AAMSRYEL + 177 DLKYNALDL + 178 HAKEKLTAL + 179IQIYKKLRTSSF + 180 LLKAEPLAF + 181 YKKLRTSSF + 182 LPFLRENDL + 183FQKLKLLSL + 184 EPVKKSRL + 185 NPNLKTLL + 186 SLIDRLVL + 187 YVKERSKAM +188 SALDHVTRL + 189 HIFLRTTL + 190 SRSMRLLLL ++ +++ 191 LINLKYLSL + 192LPMLKVLNL + 193 LSYNKYLQL + 194 EAKRHLLQV + 195 AEAVLKTLQEL ++ 196AEQTGTWKL + 197 EEAKQVLFL + 198 FELPTGAGL ++ 199 GEATLQRIY + 200GEELGFASL ++ 201 GEHTSKATL + 202 KEFNLQNMGL ++ 203 KENFAGEATL + 204KESQLPTVM ++ 205 QEVLLQTFL ++ 206 SEPINIIDAL + 207 TEATMTFKY ++ 208AEHDAVRNAL ++ 209 YEVDTVLRY +++ 210 SENIVIQVY + 211 TEKEMIQKL ++ 212AEETCAPSV + 213 TTMDQKSLW + 214 AEQPDGLIL + 215 AFITAQNHGY + 216LQEEKVPAIY + 217 NEINEKIAPSF + 218 AEGGKVPIKW +++ + 219 AENAEYLRV ++ 220KEITGFLLI +++ 221 AEERAEAKKKF + 222 NEISTFHNL + 223 SEVPVARVW + 224SESAVFHGF + 225 SEAFPSRAL +++ 226 EELLHGQLF + 227 TEHTQSQAAW + 228AEKQTPDGRKY ++ 229 KESDGFHRF + 230 AENLFRAFL + 231 AEIHTAEI +++ 232AEKDGKLTDY +++ 233 DESEKTTKSF +++ 234 EEESLLTSF +++ 235 EEFETLKEF ++ 236EEKLIIQDF ++ 237 LEMPHYSTF +++ 238 SENPETITY +++ 239 TEVLDSHIHAY ++ 240HELENHSMY + 241 REAEPIPKM + 242 FSDKELAAY + 243 RSPNNFLSY + 244RSDPVTLNV + 245 ITEKNSGLY ++ 246 YSDLHAFYY + 247 RSDPGGGGLAY + 248YSHAAGQGTGLY + 249 ALFPERITV + 250 KMILKMVQL + + +++ 251 RLASRPLLL + 252RIYNGIGVSR + + 253 KLFGTSGQK +++ 254 AVATKFVNK + 255 LPDGSRVEL ++ + +++++ 256 LPALPQQLI + + 257 SPLRGGSSL + 258 APSGTRVVQVL + 259 RPAVGHSGL +260 EEAPLVTKAF + 261 IEALLESSL + 262 MELLLVNKL + 263 QQATPGPAY ++ 264DEYLIPQQGF +++ + 265 EEVDVPIKLY + 266 ARLTPIPFGL +

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*02:01, HLA-A*24:02,HLA-A*01:01, HLA-A*03:01, HLA-B*07:02 and HLA-B*44:02 restricted TUMAPsof the invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 10a andTable 10b).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02,HLA-A*24, HLA-A*01, HLA-A*03, HLA-B*07 or HLA-B*44 leukapheresisproducts via positive selection using CD8 microbeads (Miltenyi Biotec,Bergisch-Gladbach, Germany) of healthy donors obtained from theUniversity clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 269) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.270), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+ CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity for Colorectal Cancer, Glioblastoma, GastricCancer, Hepatocellular Carcinoma, and Renal Cell Carcinoma Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T-cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 13peptides of the invention are shown in FIGS. 2 to 9 together withcorresponding negative controls. Results for 56 peptides from theinvention are summarized in Table 10a and Table 10b.

TABLE 10a in vitro immunogenicity of HLA class I peptides of the invention  Exemplary results of in vitro immunogenicity experiments conducted by the applicant forthe peptides of the invention.<20% = +; 20%-49% = ++; 50%-69% = +++;  >=70% = ++++ Seq ID No SequenceWells positive [%] 267 KTLGKLWRL ++++ 268 DYIPYVFKL ++++

TABLE 10b in vitro immunogenicity of HLA class I peptides of the invention Exemplary results of in vitro immunogenicity experiments conducted by the applicant forthe peptides of the invention. <20%  = +; 20%-49% = ++; 50%-69% = +++;  >=70% = ++++ Seq IDWells positive No Sequence [%] HLA 1 RSDPVTLDV “++” A*01 6 VLDLSTNVY“++” A*01 20 STDEITTRY “+” A*01 24 YTHHLFIFY “+” A*01 28 LTDYINANY “+”A*01 31 VTDLEMPHY “+” A*01 33 FADKIHLAY “++” A*01 37 LTDSFPLKV “+++”A*01 245 ITEKNSGLY “+++” A*01 247 RSDPGGGGLAY “+” A*01 38 VMLNSNVLL “+”A*02 42 KMMALVAEL “++++” A*02 43 GLMTIVTSL “++” A*02 50 ELMPRVYTL “+”A*02 56 FLNQIYTQL “+++” A*02 57 GLTGVIMTI “++++” A*02 60 GLPDFVKEL “++”A*02 249 ALFPERITV “++” A*02 250 KMILKMVQL “++” A*02 64 STTKLYLAK “+”A*03 66 ATYEGIQKK “+” A*03 82 TAYGSTVSK “+” A*03 94 KTYVGHPVKM “+” A*03105 GSAALYLLR “+” A*03 110 GTESGTILK “+” A*03 138 KYLSIPTVF “+” A*24 144IPEPSAQQL “+” B*07 146 SPGDKRLAA “++” B*07 148 VPDGVSKVL “+++” B*07 155FPYPYAERL “+++” B*07 156 HPQVVILSL “++” B*07 164 ISTPSEVSTPL “+” B*07170 SPMWHVQQL “++++” B*07 172 KPAHYPLIAL “+++” B*07 255 LPDGSRVEL “++++”B*07 195 AEAVLKTLQEL “+” B*44 198 FELPTGAGL “++” B*44 200 GEELGFASL “+”B*44 204 KESQLPTVM “++” B*44 205 QEVLLQTFL “+” B*44 208 AEHDAVRNAL “+”B*44 209 YEVDTVLRY “+” B*44 218 AEGGKVPIKW “++” B*44 219 AENAEYLRV “+++”B*44 220 KEITGFLLI “++” B*44 225 SEAFPSRAL “+++” B*44 232 AEKDGKLTDY “+”B*44 233 DESEKTTKSF “++++” B*44 235 EEFETLKEF “+” B*44 236 EEKLIIQDF“++++” B*44 237 LEMPHYSTF “+++” B*44 238 SENPETITY “++” B*44 239TEVLDSHIHAY “++++” B*44 264 DEYLIPQQGF “+” B*44

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizes (trifluoro acetate salt) in purities of >50%. All TUMAPs arepreferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100-fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores. Binding of HLA-class I restricted peptides to HLA-A*01:01 was ranged by peptide exchange yield:  >10% = +; >20% = ++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 1 RSDPVTLDV “+++” 2 LPSPTDSNFY “++”3 ASSTDSASYY “+++” 4 NSDLKYNAL “++” 5 SILGSDVRVPSY “++” 6 VLDLSTNVY“+++” 7 LITGDPKAAYDY “++” 8 TPVTEFSLNTY “++” 9 FITAQNHGY “++” 10ITAQNHGY “++” 11 LSAGSGPGQY “+++” 12 ITFGERFEY “+++” 13 GSTMVEHNY “++”14 YTE RDGSAMVY “+++” 15 LTDYLKNTY “+++” 16 LSLIDRLVLY “++” 17 YTDKLQHY“+++” 18 EVSNGKWLLY “+++” 19 VSNGKWLLY “+++” 20 STDEITTRY “+++” 21STDIGALMY “+++” 22 TLEQVQLYY “+++” 23 TASEDVFQY “+” 24 YTHHLFIFY “+” 25LMKEVMEHY “++” 26 EVLDSHIHAY “+” 27 LDSHIHAY “+” 28 LTDYINANY “+++” 29SVTDLEMPHY “+” 30 VLDSHIHAY “+++” 31 VTDLEMPHY “++++” 32 ATVGYFIFY “++”33 FADKIHLAY “+++” 34 ITDFNNIRY “+++” 35 FASDLLHLY “++” 36 YAAYIIHAY“+++” 37 LTDSFPLKV “+++” 242 FSDKELAAY “++++” 243 RSPNNFLSY “++” 244RSDPVTLNV “+++” 245 ITEKNSGLY “+++” 246 YSDLHAFYY “++++” 247 RSDPGGGGLAY“+++” 248 YSHAAGQGTGLY “+++”

TABLE 12 MHC class I binding scores. Binding of HLA-class I restricted peptides to HLA-A*02:01 was ranged by peptide exchange yield:  >10% = +; >20% = ++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 38 VMLNSNVLL “++++” 39 YLLPSVVLL“++++” 40 KIDDIWNLEV “+++” 41 SLQDTKITL “+++” 42 KMMALVAEL “++++” 43GLMTIVTSL “++++” 44 SQTGFVVLV “++” 45 KLLDEVTYL “+++” 46 VLITGLPLI“++++” 47 YQDSWFQQL “+++” 49 NLASRPYSL “++” 50 ELMPRVYTL “+++” 51ALAAELNQL “+++” 52 YVSSGEMMV “+++” 53 LLMTSLTES “+++” 54 YLPPTDPRMSV“++++” 55 RLWQIQHHL “+++” 56 FLNQIYTQL “++++” 57 GLTGVIMTI “++++” 59KLHEIYIQA “++++” 60 GLPDFVKEL “++++” 61 RLFGLFLNNV “++++” 249 ALFPERITV“++++” 250 KMILKMVQL “+” 251 RLASRPLLL “+++”

TABLE 13 MHC class I binding scores. Binding of HLA-class I restricted peptides to HLA-A*03:01 was ranged by peptide exchange yield:  >10% =+; >20% = ++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 62 GSYSALLAKK “+++” 63 KVLGPNGLLK“++” 64 STTKLYLAK “++” 65 VLGPNGLLK “++” 66 ATYEGIQKK “++” 67 ATALSLSNK“+++” 68 ATAYGSTVSK “++” 70 ATWSASLKNK “+++” 71 KLGNNPVSK “++” 73KSFDRHFEK “+++” 74 QLYSKFLLK “+++” 75 QVPTFTIPK “++” 76 SAFGYVFPK “+++”77 SSASLAHMK “++” 78 STKSTSPPK “++” 79 STNNEGNLK “++” 80 STSHHLVSR “++”81 SVKLQGTSK “++” 82 TAYGSTVSK “+++” 84 TVASLHTEK “++” 85 KMAAWPFSR“+++” 86 KTPSGALHRK “++” 87 SSYSRSSAVK “+++” 88 MLLQQPLIY “++” 89KITDFGLAK “++” 90 GSRLGKYYVK “+” 92 AVLDLGSLLAK “+++” 93 ALDKPGKSK “++”94 KTYVGHPVKM “+++” 95 RLFESSFHY “+++” 96 FSLAGALNAGFK “++” 97 RMPPPLPTR“++” 98 KLYPTYSTK “++” 99 ATMQSKLIQK “+++” 100 ALLGVIIAK “++” 101GVIIAKKYFFK “++” 102 IIAKKYFFK “++” 104 STQDTLLIK “++” 105 GSAALYLLR“++” 106 RLSPNDQYK “++” 109 KIIAPLVTR “++” 110 GTESGT ILK “++” 111KIKEHVRSK “++” 112 KMMADYGIRK “+++” 252 RIYNGIGVSR “+++” 253 KLFGTSGQK“+++” 254 AVATKFVNK “++”

TABLE 14 MHC class I binding scores. Binding of HLA-class I restricted peptides to HLA-A*24:02was ranged by peptide exchange yield:  >10% = +; >20% = ++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 113 VWAKILSAF “+++” 114 KFLDSNIKF“+++” 115 YFEEAANFL “++” 116 LVLDYSKDYNHW “+” 117 NFLPPIIARF “++++” 118TYISKTIAL “+++” 120 MYAKEFDLL “+++” 121 SYIEKVRFL “+++” 122 KLYGMPTDFGF“++” 123 RQYLAINQI “+” 125 IYGPKYIHPSF “++++” 126 TFQDKTLNF “+++” 127IFINLSPEF “++++” 128 SYTKVEARL “+++” 129 VFLNQIYTQL “++” 130 VYGDGHYLTF“+++” 133 SFDSNLLSF “+” 134 TYLTGRQF “++” 135 VIAPIISNF “+++” 136EYNNIQHLF “+++” 137 KYLSLSNSF “++++” 138 KYLSIPTVF “++++” 139 PYASLASELF“++++” 140 KYLSIPAVF “++++” 141 KYLSIPAVFF “++++”

TABLE 15 MHC class I binding scores. Binding of HLA-class I restricted peptides to HLA-B*07:02was ranged by peptide exchange yield:  >10% = +; >20% =++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 143 FELPTGAGLQL “++” 144 IPEPSAQQL“++” 145 RVPSYTLIL “+++” 146 SPGDKRLAA “+++” 147 SPIKVPLLL “+++” 148VPDGVSKVL “++” 149 YPLTGDTRL “++” 150 KPSSKALGTSL “+++” 151 VVHPRTLLL“++” 152 IPSRLLAIL “+++” 153 APAAVPSAPA “+++” 154 GPGTRLSL “++” 155FPYPYAERL “++” 156 HPQVVILSL “+++” 157 SPSPGKDPTL “++” 158 VPERGEPEL“++” 159 FPAHPSLLL “++” 160 RPAPADSAL “+++” 161 NPYEGRVEV “++” 162MPMISIPRV “++” 163 RPASSLRP “++” 164 ISTPSEVSTPL “++” 165 TPIAKVSEL“+++” 166 HDPDVGSNSL “++” 167 YPSEVEHMF “++” 168 IPTDKLLVI “++” 169FPTEVTPHAF “++” 170 SPMWHVQQL “+++” 171 APKLFAVAF “+++” 172 KPAHYPLIAL“+++” 173 MVPSAGQLALF “++” 174 VPSLQRLML “+++” 175 HPIETLVDIF “++” 255LPDGSRVEL “++” 256 LPALPQQLI “+++” 257 SPLRGGSSL “++” 258 APSGTRVVQVL“++” 259 RPAVGHSGL “++”

TABLE 16  MHC class I binding scores. Binding of HLA-class I restricted peptides to HLA-B*44:02was ranged by peptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++Seq ID No Sequence Peptide exchange 195 AEAVLKTLQEL “+++” 196 AEQTGTWKL“++” 197 EEAKQVLFL “++” 198 FELPTGAGL “++” 199 GEATLQRIY “+” 200GEELGFASL “++” 201 GEHTSKATL “++” 202 KEFNLQNMGL “+++” 203 KENFAGEATL“++” 204 KESQLPTVM “++” 205 QEVLLQTFL “++++” 206 SEPINIIDAL “++” 207TEATMTFKY “+++” 208 AEHDAVRNAL “+++” 209 YEVDTVLRY “++” 210 SENIVIQVY“++” 211 TEKEMIQKL “++” 212 AEETCAPSV “++” 213 TTMDQKSLW “+” 214AEQPDGLIL “++” 216 LQEEKVPAIY “+” 217 NEINEKIAPSF “+++” 218 AEGGKVPIKW“+++” 219 AENAEYLRV “++” 220 KEITGFLLI “+++” 221 AEERAEAKKKF “++” 222NEISTFHNL “+++” 223 SEVPVARVW “++++” 224 SESAVFHGF “+++” 225 SEAFPSRAL“+++” 226 EELLHGQLF “++” 227 TEHTQSQAAW “+++” 228 AEKQTPDGRKY “++” 229KESDGFHRF “+++” 230 AENLFRAFL “+++” 231 AEIHTAEI “++” 232 AEKDGKLTDY“++” 233 DESEKTTKSF “++” 234 EEESLLTSF “+++” 235 EEFETLKEF “++” 236EEKLIIQDF “++” 237 LEMPHYSTF “++++” 238 SENPETITY “+++” 239 TEVLDSHIHAY“++” 240 HELENHSMY “++” 241 REAEPIPKM “++” 260 EEAPLVTKAF “++” 261IEALLESSL “++” 262 MELLLVNKL “+++” 263 QQATPGPAY “++” 264 DEYLIPQQGF“+++” 265 EEVDVPIKLY “++” 266 ARLTPIPFGL “+”

Example 6

Peptide-MHC Class I Stability

The peptide-MHC stability for HLA-B*08:01 peptides was performed byImmunAware (Copenhagen, Denmark). The data were obtained using aproximity based, homogenous, real-time assay to measure the dissociationof peptides from HLA class I molecules. First human recombinantHLA-B*08:01 and b2m were expressed in E. coli and purified in a seriesof liquid chromatography based steps (Ferre et al., 2003; Ostergaard etal., 2001). Afterwards, the stability of a peptide-MHC complex (pMHC)can be determined by measuring the amount of b2m associated with the MHCheavy chain over time at 37° C. (Harndahl et al., 2012). The stabilityof each pMHC, expressed as the half life of b2m associated with therespective heavy chain, was calculated by fitting the data to aone-phase dissociation equation.

The pMHC stability were measured in three independent experiments andthe peptides in question, for HLA-B*08:01, were found to span the rangefrom weak-binders (+) to very stable binders (++++). The mean half-life(T1/2) is shown in Table 17.

TABLE 17 Mean half-life (T1/2) based on three individualmeasurements. T1/2 > 2 h = +; T1/2 > 4 h = ++;T1/2 > 6 h = +++; T1/2 > 10 h = ++++ Mean Half-life Seq ID No Sequence(T1/2) 176 AAMSRYEL ++ 177 DLKYNALDL +++ 178 HAKEKLTAL +++ 180 LLKAEPLAF++ 181 YKKLRTSSF ++ 182 LPFLRENDL ++ 183 FQKLKLLSL + 184 EPVKKSRL +++185 NPNLKTLL ++ 187 YVKERSKAM +++ 189 HIFLRTTL ++ 192 LPMLKVLNL + 193LSYNKYLQL ++ 194 EAKRHLLQV ++

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The invention claimed is:
 1. A method of treating a patient who hascancer that presents a peptide consisting of the amino acid sequence ofSEQ ID NO: 42 in a complex with an MHC class I molecule on the surfaceof cancer cells, comprising administering to said patient a compositioncomprising a population of activated CD8+ cytotoxic T cells that bind apeptide consisting of the amino acid sequence of SEQ ID NO: 42 in acomplex with an MHC class I molecule, wherein said cancer is selectedfrom the group consisting of renal cell carcinoma, glioblastoma, gastriccancer, hepatocellular carcinoma, and ovarian cancer.
 2. The method ofclaim 1, wherein the CD8+ cytotoxic T cells are autologous to thepatient.
 3. The method of claim 1, wherein the CD8+ cytotoxic T cellsare obtained from a healthy donor.
 4. The method of claim 1, wherein theCD8+ cytotoxic T cells are obtained from tumor infiltrating lymphocytesor peripheral blood mononuclear cells.
 5. The method of claim 1, whereinthe activated CD8+ cytotoxic T cells are expanded in vitro.
 6. Themethod of claim 1, wherein the composition further comprises anadjuvant.
 7. The method of claim 6, wherein the adjuvant is selectedfrom imiquimod, GM-CSF, poly-(ICLC), interleukin (IL)-1, IL-2, IL-4,IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
 8. The method of claim 1,wherein the activated CD8+ cytotoxic T cells are produced by contactingCD8+ cytotoxic T cells with an antigen presenting cell that expressesthe peptide in a complex with an MHC class I molecule on the surface ofthe antigen presenting cell, for a period of time sufficient to activatesaid CD8+ cytotoxic T cell.
 9. The method of claim 8, wherein theantigen presenting cell is infected with a recombinant virus expressingthe peptide.
 10. The method of claim 1, wherein the cancer is renal cellcarcinoma.
 11. The method of claim 1, wherein the cancer isglioblastoma.
 12. The method of claim 1, wherein the cancer is gastriccancer.
 13. The method of claim 1, wherein the cancer is hepatocellularcarcinoma.
 14. A method of eliciting an immune response in a patient whohas cancer that presents a peptide consisting of the amino acid sequenceof SEQ ID NO: 42 in a complex with an MHC class I molecule on thesurface of cancer cells, comprising administering to said patient acomposition comprising a population of activated CD8+ cytotoxic T cellsthat bind a peptide consisting of the amino acid sequence of SEQ ID NO:42 in a complex with an MHC class I molecule, wherein said cancer isselected from the group consisting of renal cell carcinoma,glioblastoma, gastric cancer, hepatocellular carcinoma, and ovariancancer.
 15. The method of claim 14, wherein the activated CD8+ cytotoxicT cells are produced by contacting CD8+ cytotoxic T cells with anantigen presenting cell that expresses the peptide in a complex with anMHC class I molecule on the surface of the antigen presenting cell, fora period of time sufficient to activate said CD8+ cytotoxic T cell. 16.The method of claim 14, wherein the cancer is renal cell carcinoma. 17.The method of claim 14, wherein the cancer is ovarian cancer.
 18. Themethod of claim 14, wherein the cancer is gastric cancer.
 19. The methodof claim 14, wherein the cancer is hepatocellular carcinoma.
 20. Themethod of claim 1, further comprising administering to said patientcyclophosphamide, sunitinib, bevacizumab, or sildenafil.